Rotation-dependent transcriptional sequencing systems and methods of using

ABSTRACT

Provided herein are rotation-dependent transcriptional sequencing methods and systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of, and claims the benefit ofpriority under 35 U.S.C. §120 to U.S. application Ser. No. 13/403,645filed Feb. 23, 2012, issued as U.S. Pat. No. 8,574,840 on Nov. 5, 2013,which claims the benefit of priority under 35 U.S.C. §119(e) to U.S.Application No. 61/463,850, filed on Feb. 23, 2011, and U.S. ApplicationNo. 61/574,270, filed on Jul. 30, 2011.

TECHNICAL FIELD

This disclosure generally relates to nucleic acid sequencing systems andmethods and compositions that can be used in such systems and methods.

BACKGROUND

Several techniques are currently available for detection and typing ofbacterial and viral pathogens. This includes methods employing:

-   -   1) indirect determination of genetic sequence including        species/strain-specific PCR, repetitive sequence-based PCR        (rep-PCR), pulse-field gel electrophoresis (PFGE), and optical        DNA mapping,    -   2) direct determination of the sequence by either multi-locus        sequence typing (MLST) or whole bacterial genome sequencing, or    -   3) a combination of the above such as determination of the base        composition of PCR products by mass spectrometry.

The first group of techniques (Cepheid, PathoGenetix, Inc., OpGen, Inc.)has lower resolution and discrimination power compared to the secondgroup of techniques and is limited by the small number of conservedgenomic regions interrogated. The techniques in the second group,however, have been prohibitively expensive and provide only lowthroughput, although, with the introduction of 2^(nd) (SOLiD and PGM byLife Technologies/Ion Torrent, Illumina, Roche 454, Complete Genomics)and 3^(rd) (Pacific Biosciences, Helicos) generation high throughputsequencing techniques, the per sample cost is trending below $10,000.Moreover, the currently available sequencing technologies suffer fromeither complex sample preparation and DNA cluster generation (SOLiD andIonTorrent by Life Technologies, Roche 454, Complete Genomics,Illumina), short read length (Helicos, SOLiD, Illumina), or high errorrate (Pacific Biosciences). Additionally, the currently availablesingle-molecule sequencing instruments (Pacific Biosciences and Helicos)are bulky, very expensive, and require highly trained personnel tooperate. The third group of techniques (Ibis Biosciences Inc.) is ableto determine the nucleotide composition of only relatively shortsequences of PCR products and suffers from all limitations ofconventional PCR.

In contrast to currently available single molecule technologies(Helicos, Pacific Biosciences), the rotation-dependent transcriptionalsequencing described herein does not require development of mutantpolymerases capable of incorporating modified nucleotides, expensivelabeled nucleotides, or lasers and costly high-speed cameras. Thus, therotation-dependent transcriptional sequencing described herein can beintegrated into inexpensive portable point-of-care systems.

In addition, the rotation-dependent transcriptional sequencing describedherein allows for ultimate flexibility and fast reconfiguration;permitting rapid response to unforeseen endemic threats, emergingdiseases, pandemics and new bioterror threats by simply updating theplatform-associated nucleic acid database and software without anychange of the reagents. This is in contrast to numerous diagnosticplatforms exploiting PCR, where significant time is required for assayreconfiguration and validation before platform redeployment to addressany new targets. This is also in contrast to non-sequencingsingle-molecule Genome Sequence Scanning platform using Direct LinearAnalysis (DLA) technology (PathoGenetiX), which relies upon a spatialpattern of tags separated by at least 3 kb and makes this technologyinsensitive to sub-kb insertions or deletions as well assingle-nucleotide variances.

Furthermore, the rotation-dependent transcriptional sequencing describedherein allows for ultimate multiplexing capability. While PCR- andmicroarray-based methods are limited by the detection of only knowninfectious agent(s) and cannot identify variants that are mutated orbioengineered (i.e. with a single nucleotide difference), therotation-dependent transcriptional sequencing described herein is, in asense, “target agnostic,” as the methods decode primary structure of anyand all DNA molecules in or extracted from the specimen, and, thus, iscapable of detecting and identifying thousands of known or unknown(e.g., genetically-modified) targets simultaneously. Such an inherited“broadband” multiplexing capability provided by the systems and methodsdescribed herein is in contrast to approaches employing PCR that requirespecific sets of reagents (primers and probes) for detection of eachpathogen. Additionally, PCR-based technologies are limited by the numberof assays allowed in multiplexed reactions simply due to the nature ofPCR, or by the need to split the sample (i.e., containing the targetnucleic acids) between multiple reactions, thereby compromising thesensitivity of detection and the accuracy of quantification.

SUMMARY

Rotation-dependent transcriptional sequencing relies upon the RNApolymerase being immobilized relative to the solid surface. As aconsequence of transcription, the RNA polymerase exerts torque on thenucleic acid, which, in turn, manifests itself as rotation of a tagattached to the nucleic acid.

In one aspect, a method of determining the sequence of a target nucleicacid molecule is provided. Such a method generally includes contactingan RNA polymerase with a target nucleic acid molecule under sequencingconditions, detecting the rotational pattern of the rotation tag, andrepeating the contacting and detecting steps a plurality of times.Typically, sequencing conditions include the presence of at least onenucleoside triphosphate, and the RNA polymerase is immobilized on asolid substrate, where the target nucleic acid molecule comprises arotation tag. The sequence of the target nucleic acid molecules isbased, sequentially, on the presence or absence of a change in therotational pattern in the presence of the at least one nucleosidetriphosphate.

In some embodiments, the RNA polymerase is a bacteriophage RNApolymerase (e.g., a T7 RNA polymerase, a T3 RNA polymerase). In someembodiments, the RNA polymerase is a bacterial RNA polymerase (e.g., E.coli RNA polymerase). In some embodiments, the RNA polymerase isimmobilized on the solid surface via a His-tag. Representative targetnucleic acid molecules can be prokaryotic, bacterial, archaeal, andeukaryotic. Target nucleic acid molecules typically are double-stranded,and can be comprised within a biological sample. The target nucleic acidmolecule further can include a RNA polymerase promoter sequence.

In some embodiments, the rotation tag includes a first tag and a secondtag. For example, in some embodiments, the first tag is magnetic. Arepresentative solid substrate is made from glass. Other representativesolid substrates include a CMOS or CCD. In some embodiments, thesequencing conditions include the presence of a single nucleosidetriphosphate; in some embodiments, the sequencing conditions include thepresence of four nucleoside triphosphates, where a first nucleosidetriphosphate of the four nucleoside triphosphates is present in arate-limiting amount.

In some embodiments, the detecting step includes projecting light ontothe rotation tag. In some embodiments, the detecting step furtherincludes observing the rotational pattern via a microscope. In someembodiments, the detecting step further includes capturing therotational pattern on a CMOS or CCD. In some embodiments, the detectingstep includes capturing the rotational pattern as a magnetic image. Insome embodiments, the magnetic image is captured on a GMR sensor or aMRAM array. In some embodiments, the detecting step includes capturingthe rotational pattern as an electric field. In some embodiments, theimage is captured on a RAM sensor.

Such methods also can include applying a directional force on the targetnucleic acid molecules. For example, directional force can be producedusing a magnet, or using flow or pressure.

In another aspect, a method of determining the sequence of a targetnucleic acid molecule is provided. Such a method typically includesproviding a solid substrate onto which RNA polymerase is immobilized;contacting the RNA polymerase with the target nucleic acid moleculeunder first sequencing conditions, wherein the target nucleic acidmolecule comprises a rotation tag, wherein the first sequencingconditions comprise the presence of four nucleoside triphosphates, wherea first nucleoside triphosphate of the four nucleoside triphosphates ispresent in a rate-limiting amount; detecting the rotational pattern ofthe rotation tag under the first sequencing conditions; and determiningpositional information of the first nucleoside triphosphate along thetarget nucleic acid molecule based on a change in the rotationalpattern.

Such a method can further include providing a solid substrate onto whichRNA polymerase is immobilized; contacting the RNA polymerase with thetarget nucleic acid molecule comprising the rotation tag under secondsequencing conditions, wherein the second sequencing conditions comprisethe presence of four nucleoside triphosphates, where a second nucleosidetriphosphate of the four nucleoside triphosphates is present in arate-limiting amount; detecting the rotational pattern of the rotationtag under the second sequencing conditions; and determining positionalinformation of the second nucleoside triphosphate along the targetnucleic acid molecule based on a change in the rotational pattern.

In some embodiments, the contacting and detecting steps under the secondsequencing conditions are performed simultaneously with the contactingand detecting steps under the first sequencing conditions. In someembodiments, the contacting and detecting steps under the secondsequencing conditions are performed sequentially before or after thecontacting and detecting steps under the first sequencing conditions.

Such methods also can include providing a solid substrate onto which RNApolymerase is immobilized; contacting the RNA polymerase with the targetnucleic acid molecule comprising the rotation tag under third sequencingconditions, wherein the third sequencing conditions comprise thepresence of four nucleoside triphosphates, where a third nucleosidetriphosphate of the four nucleoside triphosphates is present in arate-limiting amount; detecting the rotational pattern of the rotationtag under the third sequencing conditions; and determining positionalinformation of the third nucleoside triphosphate along the targetnucleic acid molecule based on a change in the rotational pattern. Suchmethods can further include determining the sequence of the targetnucleic acid molecule from the positional information for the first,second and third nucleoside triphosphates within the target nucleic acidmolecule.

Such methods further can include providing a solid substrate onto whichRNA polymerase is immobilized; contacting the RNA polymerase with thetarget nucleic acid molecule comprising the rotation tag under fourthsequencing conditions, wherein the fourth sequencing conditions comprisethe presence of four nucleoside triphosphates, where a fourth nucleosidetriphosphate of the four nucleoside triphosphates is present in arate-limiting amount; detecting the rotational pattern of the rotationtag under the fourth sequencing conditions; and determining positionalinformation of the fourth nucleoside triphosphate along the targetnucleic acid molecule based on a change in the rotational pattern.

In some embodiments, the solid surface is a glass slide. Such a glassslide can be coated with Copper and PEG. In some embodiments, the RNApolymerase is a T7 RNA polymerase. In some embodiments, the T7 RNApolymerase is immobilized on the solid substrate via a His-tag.

In another aspect, a method of determining the sequence of a targetnucleic acid molecule is provided. Such a method typically includesproviding a solid substrate onto which one or more RNA polymerases areimmobilized; contacting the one or more RNA polymerases with the targetnucleic acid molecule under first sequencing conditions, wherein thetarget nucleic acid molecule comprises a rotation tag, wherein the firstsequencing conditions comprise the presence of a first of fournucleoside triphosphates; and detecting, under the first sequencingconditions, whether a change in the rotational pattern occurs. If achange in the rotational pattern occurs, the method further comprisesrepeating the contacting step and subsequent steps under the firstsequencing conditions, while, if a change in the rotational pattern doesnot occur, the method further comprises repeating the contacting stepand subsequent steps under second sequencing conditions, wherein thesecond sequencing conditions comprise the presence of a second of fournucleoside triphosphates Similarly, if a change in the rotationalpattern occurs, the method further comprises repeating the contactingstep and subsequent steps under the first sequencing conditions, while,if a change in the rotational pattern does not occur, the method furthercomprises repeating the contacting step and subsequent steps under thirdsequencing conditions, wherein the third sequencing conditions comprisethe presence of a third of four nucleoside triphosphates. The sequenceof the target nucleic acid molecule can be obtained based, sequentially,on the occurrence of a change in the rotational pattern under the first,second, or third sequencing conditions.

In still another aspect, an article of manufacture is provided. Articlesof manufacture typically include a solid substrate onto which aplurality of RNA polymerase enzymes are immobilized. In someembodiments, the solid substrate is coated with copper and PEG; inanother embodiment, the solid substrate is coated with nickel and PEG.Alternatively, the solid substrate can be coated with Ni-NTA. In someembodiments, the solid substrate is a CMOS or CCD.

In some embodiments, an article of manufacture further includes arotation tag. A rotation tag can include a non-spherical tag, or aspherical tag having a non-uniform surface that can be distinguishedoptically. An article of manufacture also can include T7 RNA polymerasepromoter sequences and/or biotinylated nucleic acid tether sequences.Articles of manufacture as described herein also can include one or morenucleoside triphosphates.

In some embodiments, the article of manufacture further includesinstructions for: identifying rotation of the rotational tag relative toan axis through the magnetic reference tag; compiling a sequence of atarget nucleic acid molecule based on the rotation and the presence of anucleoside triphosphate; or applying a magnetic force. Such instructionscan be provided in electronic form.

In yet another aspect, an apparatus for single-base sequencing of targetnucleic acid molecules is provided. Such an apparatus typically includesa Sequencing Module, wherein the Sequencing Module includes a receptaclefor receiving a solid substrate, wherein the solid substrate comprises aplurality of RNA polymerases immobilized thereon; a source for providingdirectional force, wherein the directional force is sufficient and in adirection such that tension is applied to target nucleic acid moleculesbeing transcribed by the plurality of RNA polymerases immobilized on thesolid surface; a light source for projecting light onto a rotation tagbound to target nucleic acid molecules being transcribed by theplurality of RNA polymerases immobilized on the solid surface; andoptics for detecting a rotational pattern of a rotation tag bound totarget nucleic acid molecules being transcribed by the plurality of RNApolymerases immobilized on the solid surface.

Such an apparatus further can include a computer processor, and/orfluidics for containing and transporting reagents and buffers involvedin sequencing nucleic acids. Representative reagents nucleosidetriphosphates and representative buffers can be a wash buffer. In someembodiments, the source for providing directional force can be a magnetor a flow of liquid. In some embodiments, the optics comprises amicroscope, and further can includes a camera.

Such an apparatus further can include a Sample Preparation Module,wherein the Sample Preparation Module includes a receptacle forreceiving a biological sample; and fluidics for containing andtransporting reagents and buffers involved in isolating and preparingnucleic acids for sequencing. Representative reagents include cell lysisreagents and/or cleavage enzymes, while representative buffers includelysis buffer and/or wash buffer.

Such an apparatus further can include a Template Finishing Module,wherein the Template Finishing Module includes fluidics for containingand transporting reagents and buffers involved in attaching RNApolymerase promoter sequences and rotation tags to nucleic acidmolecules. Representative reagents include ligase enzyme, a molecularmotor-binding sequence, a magnetic tag, and/or a tether, whilerepresentative buffers include ligase buffer, magnetic referencetag-binding buffer, and/or rotational tag-binding buffer.

In yet another aspect, a method of determining the sequence of a targetnucleic acid molecule is provided, where the target nucleic acidmolecules include a rotation tag, and where the sequence of the targetnucleic acid molecule is based upon data obtained during transcriptionof the target nucleic acid molecule. Such a method generally includesreceiving a first datum for a first position of the target nucleic acidmolecule, wherein the first datum indicates the presence or absence ofrotation and/or the length of time between rotations of the rotationtag; receiving a second datum for the first position of the targetnucleic acid molecule, wherein the second datum indicates the presenceand/or amount of one or more nucleoside triphosphates available duringtranscription; receiving another first datum and another second datumfor a second position of the target nucleic acid molecule; receiving yetanother first datum and yet another second datum for a third position ofthe target nucleic acid molecule; repeating the receiving steps of thefirst datum and the second datum for a fourth and subsequent positionsof the target nucleic acid molecule; and determining a sequence of thetarget nucleic acid molecule based on the first datum and second datumreceived for each position. In some embodiments, the first datum and thesecond datum is recorded as a nucleotide at an indicated position.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the systems, methods and compositions of matter belong.Although systems, methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the systems,methods and compositions of matter, suitable systems, methods andmaterials are described below. In addition, the systems, materials,methods, and examples are illustrative only and not intended to belimiting. Any publications, patent applications, patents, and otherreferences mentioned below are incorporated by reference in theirentirety.

DESCRIPTION OF DRAWINGS

FIG. 1 shows an embodiment of a single-molecule rotation-dependenttranscriptional sequencing complex (FIG. 1A) and an embodiment ofon-chip (FIG. 1B) rotation-dependent transcriptional sequencingcomplexes, as described herein.

FIG. 2A is an image of magnetic rotation tags using a 20× waterobjective. In this image, the FOV is limited by the detector and not bythe objective. In this image, the magnetic rotation tags have a diameterof 2.7 microns. FIG. 2B is an image of magnetic rotation tags undertension (here, in the presence of a magnetic force), which shows,compared to FIG. 2A, that the magnetic rotation tags have moved“upwards” due to application of the magnet. The double-images arecreated due to the presence of two light sources illuminating therotation tag from above at two different angles. Therefore, whenrotation tags move out of the focal plane, the two “shadows” separate.This method can be used to determine how uniform the tension is in theplane of the sample and in different z-axis locations out of the plane.The rotation tags that are still in-focus in FIG. 2B are likely clumpedon the solid surface. FIG. 2C is a photograph of an array of rotationtags tethered by His-tagged RNA polymerase and a 5.1 Kb DNA template ona passivated solid surface.

FIG. 3 are graphs showing two modes of nucleic acid sequencing describedherein: Panel A shows an asynchronous, real-time “nucleotide pattern”sequencing strategy, where a limited concentration of a singlenucleoside triphosphate (guanine (G) in this Panel) causes thepolymerase to pause when incorporating G nucleotides into the nascentstrand. Panel B shows a synchronous sequencing strategy, where a“base-by-base” introduction of nucleoside triphosphates results in acontinuous decoding of the nucleotide sequence. The hypotheticaltranscription product (SEQ ID NO:5) is shown along the slope of the lineand the corresponding DNA sequence (SEQ ID NO:6) of the hypotheticaltranscription product is shown at the bottom of the graph.

FIG. 4A are exemplary panels showing the results from four differentflow cells, each having a different nucleoside triphosphate present in alimiting amount as indicated in each panel. FIG. 4B shows the nucleicacid sequence of the hypothetical transcription product (SEQ ID NOs: 7(top) and 8 (bottom)) compiled from the four flow cells.

FIG. 5 is a schematic showing one embodiment of a flow cell as describedherein. For example, a flow cells can include a laser-cut PressureSensitive Adhesive (PSA) film of approximately 50 micron thickness laidon top of an OmniVision sensor (OV14810; after removal of the window andcoating with SiN). This configuration results in a flow cell on top of aCMOS chip. A glass or polymeric slide, having holes for microfluidicfeed-through tubes, is then applied.

FIG. 6A is a photograph and FIG. 6B is a line drawing showing aprototypical solid surface in which fibers are used to deliver light anda microscope objective is used for imaging. A solid surface slidecontaining rotation-tagged nucleic acids and RNA polymerases is shownpositioned below the fibers and above the objective.

FIG. 7 shows one embodiment of a rotation-dependent transcriptionalsequencing system as described herein. The schematic shows a tensionsource, a source of illumination, and four flow cells on top of solidsurface chips with in- and out-fluidics.

FIG. 8A is a photograph and FIG. 8B is a line drawing showing oneembodiment of a rotation-dependent transcriptional sequencing system.This embodiment uses a 20× water-objective, “super-lens” from Olympusthat provides more than 1 mm FOV with NA=0.9, which is high resolutionwith a large field of view. The solid surface, light delivery andtension source (e.g., a magnet) are also shown.

FIG. 9A is a photograph and FIG. 9B is a line drawing showing anotherembodiment of a rotation-dependent transcriptional sequencing system. Inthis embodiment, a compact optical system is shown with an AF opticalresolution target, illumination fibers, a 20× Mitutoyo objective, tubelens and camera. A disk magnet is also visible above the target but outof the way for this image. For 1 micron resolution at objective space,one or more cameras having a pixel size greater than 20 micron can beused.

FIG. 10 shows that rotation of a rotation tag (e.g., a doubletcomprising a first tag and a second tag) can be detected even when usinglow resolution (i.e. 10×10 pixels per bead imaged through an objectiveor via the on-chip method). Further reduction to 5×5 pixels is possibleusing more advanced algorithms. Furthermore, if a tapered bundle isused, then the resolution increases for each bead that is located on topof the bundle cores by a factor related to the size of the cores and thesize of each pixel at the other side of the bundle, as well as thenumber of pixels on the detector the cores span.

FIG. 11A is a flow diagram illustrating an example analysis of therotational pattern of a rotation tag, and FIG. 11B is a flow diagramillustrating an example process for determining the sequence of a targetnucleic acid molecule.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The present disclosure describes a single molecule sequencing system inwhich many of the constraints of existing single molecule system arerelaxed, including complexity, cost, scalability and, ultimately, longerread lengths, higher throughput and enhanced accuracy. The real time,single molecule sequencing method and system described herein cansequence thousands of nucleotides in a very short time with highaccuracy due to highly processive transcriptional machinery and simpleoptical and imaging systems.

The advantages of the present system are numerous. For example, doublestranded nucleic acid is used as the template, which minimizes andlimits the requirements for sample preparation. In addition, labelednucleotides are not required, since very simple imaging methods can beused (e.g., CMOS), which significantly reduces the cost. Also, wild typeRNA polymerase enzymes can be used; no special modifications to theenzyme are necessary, and the surface chemistry and enzymeimmobilization technologies are routine. The present systems and methodsare suitable for homopolymeric sequences, since rotation is the same foreach nucleotide and, thus, is cumulative over multiple nucleotides. Thepresent systems and methods also are readily adaptable for highthroughput sequencing.

Overview of Rotation-Dependent Transcriptional Sequencing

Rotation-dependent transcriptional sequencing relies upon transcriptionof target nucleic acid molecules by RNA polymerase. The RNA polymeraseis immobilized on a solid surface, and a rotation tag is bound to thetarget nucleic acid molecules. During transcription, RNA polymeraseestablishes a transcription bubble in the template nucleic acid thatcontains within it an RNA:DNA hybrid of approximately 8 bases. As theRNA polymerase advances along the double-stranded nucleic acid template,it must unwind the helix at the leading edge of the bubble and reannealthe strands at the trailing edge. The torque produced as a result of theunwinding of the double-stranded helix results in rotation of thetemplate nucleic acid relative to the RNA polymerase of about 36° pernucleotide incorporated. Therefore, when the RNA polymerase isimmobilized on a solid surface and a rotation tag is attached to thetemplate nucleic acid, the rotation of the template nucleic acid can beobserved and is indicative of transcriptional activity (i.e.,incorporation of a nucleoside triphosphate) by the enzyme.

As described herein, the sequence of target nucleic acid molecules aredetermined or obtained based upon changes in the rotational pattern of arotation tag. Also as described herein, detecting the presence orabsence of rotation of the nucleic acid can be done, for example, usingillumination of the rotation tag bound to the target nucleic acidmolecules. The rotational sequencing methods described herein can bescaled up for whole genome sequencing using an array of RNA polymeraseenzymes and any number of routine imaging methods suitable for capturingthe rotation of the rotation tag.

FIG. 1 shows a single-molecule sequencing embodiment (FIG. 1A) and anon-chip array embodiment (FIG. 1B) of the rotation-dependenttranscriptional sequencing complex described herein. Therotation-dependent transcriptional sequencing complex described hereincan be used to determine the sequence of a target nucleic acid molecule10. In some embodiments, RNA polymerase 20, immobilized on a solidsurface 30 or on a chip 80, is contacted with a target nucleic acidmolecule 10 (i.e., a nucleic acid molecule having an unknown sequence).According to the methods described herein, the target nucleic acidmolecule 10 includes a rotation tag 40. Under particular sequencingconditions, which include the presence of at least one nucleosidetriphosphate, changes in the movement and/or velocity of the rotationtag 40 create a rotational pattern. These steps are repeated multipletimes, and the sequence of the target nucleic acid is determined based,sequentially, on the presence or absence of a change in the rotationalpattern under the particular sequencing conditions. These features arediscussed in more detail below.

Solid Surface

For the rotation-dependent transcriptional sequencing described herein,an RNA polymerase is immobilized on a solid surface. In the embodimentsdescribed herein, a solid surface typically is made from a silica-basedglass (e.g., borosilicate glass, fused silica, or quartz). Solid surfacematerials for single molecule imaging are well known and routinely usedin the art, and the same solid surface materials used for singlemolecule imaging also can be used in an array format. However, othermaterials (e.g., polypropylene, polystyrene, silicon, silicon nitride,and other polymers or composites thereof) also can be used provided theyare suitable for use in the sequencing described herein. FIG. 1B showsan “on-chip” embodiment, where the solid surface 30 also includes thedetection system. These types of solid surfaces (e.g., CMOS and CCD) arealso known in the art and are discussed in more detail below.

Before immobilizing one or more biological molecules into a solidsurface, the solid surface generally is modified (e.g., functionalized)to receive and bind the biological molecules. Methods of functionalizingsolid surfaces for immobilizing biological enzymes are known in the art.In some embodiments, the solid surface can be functionalized with copperor nickel, while in some embodiments, the solid surface can befunctionalized with Ni-NTA (see, for example, Paik et al., 2005, Chem.Commun. (Camb), 15:1956-8) or Cu-NTA. Alternatively, metals such ascobalt or the like can be used to modify a solid surface forimmobilization.

Prior to modifying a solid surface, the solid surface can be treatedwith, for example, PEG moieties. Such strategies can be used to regulatethe density of RNA polymerases on a solid surface, and also can be usedto generate a pattern of RNA polymerases on the solid surface, such as auniform, a semi-ordered or a random array of RNA polymerases. The PEGenvironment results in minimal interactions between the enzyme and thesurface (except for the binding tag on the N- or C-terminus), andultimately results in minimal disturbance to the native conformation ofthe immobilized enzyme. In addition, surface passivation methods areknown in the art and can include, for example, treating the solidsurface with bovine serum albumin (BSA).

RNA Polymerase

The rotation-dependent transcriptional sequencing methods describedherein are based on the action of RNA polymerase during the process oftranscription and the rotational force produced on the transcribednucleic acid (see, for example, US 2007/0077575). While multi-subunitRNA polymerases (e.g., E. coli or other prokaryotic RNA polymerase orone of the eukaryotic RNA polymerases) can be used in the sequencingmethods described herein, the small, single-subunit RNA polymerases suchas those from bacteriophage are particularly suitable. Single subunitRNA polymerases or the genes encoding such enzymes can be obtained fromthe T3, T7, SP6, or K11 bacteriophages.

The bacteriophage RNA polymerases are very processive and accuratecompared to many of the multi-subunit RNA polymerases, and often producefewer deletion-insertion errors. Additionally, RNA polymerases frombacteriophage are significantly less prone to back-tracking compared tomulti-subunit counterparts such as the RNA polymerase from E. coli. RNApolymerase from several different bacteriophages has been described.Simply by way of example, the T7 RNA polymerase is made up of a singlepolypeptide having a molecular weight of 99 kDa, and the cloning andexpression of the gene encoding T7 RNA polymerase is described in U.S.Pat. No. 5,693,489. The structure of T7 RNA polymerase has been resolvedto a level of 3.3 Angstroms, with four different crystal structureshaving been solved: T7 RNA polymerase alone (uncomplexed), T7 RNApolymerase bound to a nucleic acid promoter, the entire initiationcomplex (T7 RNA polymerase bound to a nucleic acid promoter and one ormore transcription factors), and T7 RNA polymerase bound by aninhibitor.

RNA polymerases that are suitable for use in the methods describedherein typically provide rotations of the nucleic acid having durationsin the range of sub-microsecond to 100 milliseconds for every nucleotideincorporated into the transcript, but RNA polymerases that providerotations having durations in the range of 100 milliseconds up toseveral seconds per nucleotide also can be used. It would be understoodby those skilled in the art that, in order to produce the necessaryrotation, the RNA polymerase must transcribe double-stranded nucleicacid.

The density and/or distribution of RNA polymerases on a solid surfacecan be controlled or manipulated, for example, to optimize theparticular sequencing reactions being performed. As is known in the art,an array of biological molecules can be generated in a pattern. Forexample, an array of biological molecules can be randomly distributed onthe solid surface, uniformly distributed or distributed in an ordered orsemi-ordered fashion. In some embodiments, a solid surface can havegreater than 100 RNA polymerases, or greater than 1000 RNA polymerases(e.g., greater than 10,000 RNA polymerases) immobilized thereon. In someembodiments, a solid surface can have at least one RNA polymeraseimmobilized per ˜5 μm² (e.g., at least one RNA polymerase immobilizedper ˜2.5 μm², ˜1 μm², ˜0.5 μm², or ˜0.1 μm²). It would be understoodthat the density of RNA polymerases on a solid surface may depend, atleast, in part, upon the size of the target nucleic acid molecules beingsequenced as well as the particular rotation tag utilized.

While the sequencing methods described herein rely upon the rotationcreated during transcription by RNA polymerase, other molecular motorscan be used in conjunction with RNA polymerase to create rotation.Molecular motors are biological molecules that consume energy, typicallyby hydrolysis of a nucleotide triphosphate, and convert it into motionor mechanical work. Examples of molecular motors include, withoutlimitation, helicases, topoisomerases, DNA polymerases, myosin, ATPasesand GTPases. In some embodiments, a molecular motor can be immobilizedon a solid surface and transcription by RNA polymerase can occur at aposition on the target nucleic acid molecule between the molecular motorand the rotation tag. In such instances, the rotational pattern would bea result of any rotational force placed on the nucleic acid molecule bythe molecular motor combined with the rotational force placed on thenucleic acid molecule by the RNA polymerase. As an alternative to anenzymatic molecular motor, a solid state MEMS motor, for example, can beused, in conjunction with RNA polymerase, to generate rotation.

RNA polymerase can be immobilized on a solid surface using any number ofknown means. For example, in one embodiment, the RNA polymerase containsa His-tag (e.g., His tags having 4 His residues, 6 His residues, or 10His residues). A His-tag or other suitable tag can be used provided itis compatible with the surface chemistry (e.g., functionalization)discussed above.

Target Nucleic Acid Molecules

Nucleic acid molecules for rotation-dependent transcriptional sequencingcan be obtained from virtually any source including eukaryotes, bacteriaand archaea. Eukaryotic nucleic acids can be from humans or othermammals (e.g., primates, horses, cattle, dogs, cats, and rodents) ornon-mammals (e.g., birds, reptiles (e.g., snakes, turtles, alligators,etc.) and fish), while prokaryotic nucleic acids can be from bacteria(e.g., pathogenic bacteria such as, without limitation, Streptococcus,E. coli, Pseudomonas, and Salmonella) or Archaea (e.g., Crenarchaeota,and Euryarchaeota).

Nucleic acid molecules for rotation-dependent transcriptional sequencingcan be contained within any number of biological samples. Representativebiological samples include, without limitation, fluids (e.g., blood,urine, semen) and tissues (e.g., organ, skin, mucous membrane, andtumor).

As discussed herein, one of the advantages of the rotation-dependenttranscriptional sequencing methods described herein is thatdouble-stranded nucleic acid is used as the template. This reduces theneed to manipulate the sample and the nucleic acid, which is asignificant advantage, particularly when sequencing nucleic acidsgreater than 1 Kilobase (Kb; e.g., greater than 2 Kb, greater than 5 Kb,greater than 10 Kb, greater than 20 Kb, or greater than 50 Kb) inlength, since many methods used to obtain nucleic acids from biologicalsamples result in undesired cleavage, shearing or breakage of thenucleic acids. Obviously, single-stranded nucleic acids (or samplescontaining single-stranded nucleic acids) can be used in the presentmethods. However, such single-stranded nucleic acids must be convertedinto a double-stranded nucleic acid in order to exhibit rotation duringtranscription. Methods of making double-stranded nucleic acids are wellknown in the art and will depend upon the nature of the single-strandednucleic acid (e.g., DNA or RNA). Such methods typically include the useof well known DNA polymerases and/or Reverse Transcriptase enzymes.

Sample preparation will be dependent upon the source, but typically willinclude nucleic acid isolation followed by promoter ligation. Nucleicacid templates used in the sequencing methods described herein do notrequire any special preparation and, thus, standard DNA isolationmethods can be used. Finally, a promoter sequence that is recognized bythe particular RNA polymerase being used in the transcriptionalsequencing system must be ligated to the target nucleic acid molecules.Promoter sequences recognized by a large number of RNA polymerases areknown in the art and are widely used. In addition, methods of ligatingone nucleic acid molecule (e.g., a promoter sequence) to another nucleicacid molecule (e.g., a target nucleic acid molecule having an unknownsequence) are well known in the art and a number of ligase enzymes arecommercially available.

In addition, isolated nucleic acids optionally can be fragmented and, ifdesired, particular sizes can be selected or fractionated. For example,isolated nucleic acids can be fragmented using ultrasonication and, ifdesired, size-selected using routine gel electrophoresis methodology.

In addition, the target nucleic acids optionally can be circularizedinto, for example, a plasmid, so that sequencing can be performed on acircular target in a repetitive or recursive fashion.

Rotation Tags

For the rotation-dependent transcriptional sequencing methods describedherein, the target nucleic acid molecule being sequenced includes arotation tag bound thereto. Such a tag is fixed to the nucleic acid suchthat it rotates under the torque imparted on the nucleic acid by the RNApolymerase. Rotation tags can be as large as many microns (e.g., greaterthan 1 micron, 2 microns, 3 microns or 5 microns) in diameter and assmall as nanometers (e.g., about 50 nm, 100 nm, 250 nm, 500 nm, 750 nm,850 nm, or 950 nm) in diameter. As used herein, a rotation tag can benon-spherical or spherical; however, if the rotation tag is a singlesphere, it must have some non-uniform feature that can be used to detectrotation.

A non-spherical tag can include a single moiety that is not a sphere(e.g., a tapered rod, triangular, conical, or egg-shaped). In addition,a non-spherical tag can be made using two (or more) different sizespherical tags (e.g., a first tag attached to a second tag) that,together, provide an asymmetry that allows detecting of rotation. Insome embodiments, the first and second tags can be the same oressentially the same size. For example, the first tag, tethered to thetarget nucleic acid molecule, can be considered the reference tag, whilethe second tag, attached to the first tag, can be considered therotation tag. In some embodiments, the first tag, tethered to the targetnucleic acid molecule, is larger while the second tag, attached to thefirst tag, is smaller. This configuration places the second tag as faras possible from the point of rotation of the first tag, which enhancesthe resolution of the rotation under optical detection. Thus, the sizeof the smaller tag has to be large enough for detection purposes butsmall enough to minimize the hydrodynamic effects due to the size of theoverall rotation tag.

For example, in some embodiments, a rotation tag can include a largerbead (e.g., from about 1 micron up to about 3 microns in diameter) as afirst tag and a smaller bead (e.g., from about 0.5 microns up to about 1micron in diameter) as a second tag. For example, the larger bead can be0.75 or 1 micron and the smaller bead can be 0.5 micron. These sizes areadequate to resolve rotation (using, as described in more detail below,for example, an optical system that includes a 50× magnificationMitutoyo objective with numerical Aperture of 0.75 in combination with a1× tube lens and a scientific CMOS camera of 6.5 micrometer pixel sizeor smaller). In some embodiments, a first tag can be 0.75 microns indiameter and a second tag can be 0.35 microns in diameter. These sizesalso are adequate to resolve rotation (using, as described in moredetail below, for example, a 100× objective with a numerical aperture of0.9 or higher using a scientific CMOS camera).

An attachment between, e.g., a first tag and a second tag can be amechanical tether or linkage (e.g., streptavidin-biotin bond), achemical bond (e.g., amine or carboxy), a magnetic attraction, or anycombination thereof. In some embodiments, a first tag and a second tagcan be physically attached to one another through, for example, apolymerization reaction.

On the other hand, a rotation tag can be spherical provided that itsrotation can be detected. The use of a rotation tag that is sphericalreduces or eliminates the non-linear dynamics created by a non-sphericalrotation tag, and a spherical rotation tag will exhibit lowerhydrodynamic resistance during rotation than a non-spherical rotationtag. One example of a spherical tag having a non-uniform feature thatcan be used to detect rotation is a Janus bead. See, for example,Casagrande et al. (1989, Europhys. Lett. 9:251). A Janus bead refers toa spherical bead in which one hemisphere is hydrophobic and the otherhemisphere is hydrophilic, due to a nickel coating on half of thesphere. The different features of the hemispheres allows for detectingrotation, even when the rotation tag is spherical.

In some embodiments discussed herein, a rotation tag can be magnetic.Magnetic tags, spherical or non-spherical, are well known in the art andcan be in the form of magnetic beads, rods, or other magnetic moietiessuch as, without limitation, superparamagnetic particles. The entirerotation tag can be magnetic, or only a portion of the rotation tag canbe magnetic. For example, in some embodiments, only the first tag of arotation tag can be magnetic. There are a number of commercial sourcesfor magnetic tags. The following paragraphs describe a number of ways inwhich a magnetic rotation tag can be generated for use in the sequencingmethods described herein.

In some embodiments, a nanomagnetic solution (e.g., 1% w/v in toluene orxylene with Cobalt, or Fe3o4 or FePt in toluene or xylene) can beapplied to ordinary polymeric beads to make them magnetic. In someembodiments, the nanomagnetic materials can be applied to half of thesurface of the polymer bead. This results in a magnetic bead whosehalves exhibit a different index of refraction or other optical property(e.g., scattering). In some embodiments, superparamagnetic particles (orhalf of the particle) can be coated with nanosilver ink to createoptically reflecting, yet fully superparamagnetic beads. In theseembodiments, deposition and evaporation processes can be used with such“inks” (e.g., PRIMAXX) to impart particular optical properties onmagnetic particles or portions of magnetic particles. In someembodiments, superparamagnetic particles (or half of the particle) canbe coated with quantum dots to impart particular optical properties onparticles that already are magnetic. In some embodiments, magnetochromicparticles can be used as rotation tags. Within magnetochromic particles,nanomagnets form chains within polymers or encapsulated in Carbonshells. The chains of nanoparticles are aligned with respect to eachother using an external magnetic or electromagnetic field. When lightscatters from the particle, the aligned chains act as Bragg diffractiongratings and scatter light in appropriately defined directions. Themagnetochromic particles rotate inside an external field such thatintensity modulation can be monitored between the stopped particle withall chains aligned and the particle in rotation where the chains are notcompletely aligned to determine the rotation duration from a startingposition to a resting or stopping position until the next rotation.

In some embodiments, a first magnetic tag can be combined with a secondtag that can induce an electric field change onto a detector. Suchelectric tags are known in the art and can be polymeric spheres thatinclude metallic elements, are charged, or are micron-sizedradiofrequency oscillators that induce a change in the magnetic orelectric field, which can be captured on an instrument (described inmore detail below).

Simply to provide a spatial perspective and without being bound by anyparticular size or distance limitation, the bottom surface of a rotationtag, even when a 10 Kb nucleic acid molecule is being transcribed, stillmay only be 3 μm from the top of the solid surface. That is, therotation tag is very close to the solid surface even at the beginning oftranscription. At the end of transcription, the rotation tag could benearly contacting the RNA polymerase enzyme, and so separated from thetop of the solid surface by a very small distance. In other words, arotation tag that has a diameter of 2 μm will be approximately the samesize as the nucleic acid molecule being transcribed. It would beunderstood by those in the art that transcription of the target nucleicacid molecules by RNA polymerase could proceed in the oppositedirection, thereby moving the rotation tag farther away from the solidsurface during transcription.

Rotation tags can be attached to target nucleic acid molecules usingtethers. Tethers to attach rotation tags to target nucleic acidmolecules are known in the art and include, without limitation, achemical linkage (e.g., crosslinking, van der Walls or hydrogen bond) ora protein linkage (e.g., biotin-streptavidin binding pairs, digoxigeninand a recognizing antibody, hydrazine bonding or His-tagging). Forexample, in some embodiments, a rotation tag can be coated, at leastpartially, with streptavidin, while a biotinylated nucleic acid tethercan be ligated to the target nucleic acid molecules. In someembodiments, a biotin-labeled nucleic acid (e.g., about 500 base pairs(bp)) can be ligated to one end of the target nucleic acid molecules.The target nucleic acid molecules having the biotin-labeled tether thencan be combined with streptavidin-coated rotation tags. There are anumber of commercially available tags, including magnetic tags that arecoated or partially coated with various chemistries that can be used totether the target nucleic acid molecules and/or bind a second tag (e.g.,Dynal, Invitrogen, Spherotech, Kisker Inc., Bangs Laboratories Inc.).

Tension on the Nucleic Acid Molecules

In some embodiments, the rotation-dependent transcriptional sequencingmethods described herein include applying a directional force on thetarget nucleic acid molecules, which results in the target nucleic acidmolecules being placed under some amount of tension. Tension on thetarget nucleic acid molecules becomes important with longer targetnucleic acid molecules, as longer nucleic acid molecules can fold-up orcollapse on themselves. Any type of abnormal helical structure of thetarget nucleic acid molecules could dampen or mask the torquetransferred from the RNA polymerase to the rotation tag.

The directional force applied to the target nucleic acid molecules needsto be sufficient so as to maintain the double-stranded helical nature ofthe target nucleic acid molecule, particularly downstream of thetranscription complex, and particularly when the rotation tag isthousands or hundreds of thousands of nucleotides away from the RNApolymerase. However, the directional force applied to the target nucleicacid molecules can't be so strong (i.e., apply so much tension) suchthat rotation of the rotation tag is impeded in any way or the backboneof the target nucleic acid molecule breaks. Such tension on the targetnucleic acid molecules also can reduce the Brownian motion of therotation tag or other noise effects (e.g., thermofluidic noise effects),thereby increasing the accuracy of detecting the rotational pattern ofthe rotation tag.

The tension is intended to elevate the nucleic acid-tethered rotationtag up and away from the surface in a prescribed amount of force andlocation in the three-dimensional space. The direction of the tensioncan extend the target nucleic acid in essentially a 90° angle (i.e., inthe z-axis) relative to the plane of the solid surface (i.e., in thex-axis), but the directional force also can extend the target nucleicacid molecules in a direction that is more or less than 90° relative tothe plane of the solid surface. It would be understood, however, thatthe rotation tag cannot be so near the solid surface that the surface(e.g., due to surface chemistry or surface fluidic phenomena) interfereswith (e.g., changes, alters, dampens, reduces, eliminates) the torqueprofile and/or the pattern of movement of the rotation tag as a signalof RNA polymerase activity.

In some embodiments, the tension source (or the source of thedirectional force) can be a magnet. In such cases, the rotation tag or aportion of the rotation tag can be magnetic. As indicated herein,magnetic tags (e.g., beads, rods, etc.) are well known in the art. Forexample, a magnetic force can be applied that provides a uniform spatialforce in the direction of the z-axis at a magnitude of, for example,about 1 pN, to adequately stretch the target nucleic acid molecules andavoid any looping. At the same time, such magnets generate only aminiscule force in the direction of the x-axis. These features allow therotation tags to freely rotate, while stabilizing any Brownian motion ofthe rotation tags. In some embodiments, the tension source can be aresult of a directional flow of, for example, liquid (e.g., water orbuffer) or air.

The amount of tension applied to the target nucleic acid molecules canbe calibrated using standard fluidic methodology and incorporated indata acquisition and analysis process or base calling algorithms. Forexample, such a calibration can include monitoring the Brownian motionof a rotation tag, attached to a nucleic acid molecule being transcribedby a RNA polymerase, which is immobilized on the surface, at variouslocations above the surface, at various angles relative to the plane ofthe surface, and/or in different flows or magnetic fields.

Simply to provide a spatial perspective and without being bound by anyparticular size or distance limitation, in an on-chip embodiment, therotation tag can be on the order of 1 micron above the surface whentension is applied. For example, since each base is separated by 0.3 nmfrom the next base, a one Kb nucleic acid molecule that is attached atthe surface to the RNA polymerase will have the rotation tag on theother end at a distance of about 300 nm from the surface. This distancecan be varied using, without limitation, different immobilizationmethods of the RNA polymerase to the solid surface and nucleic acids(e.g., promoter sequences, tether sequences) of different lengths. Therotation-dependent transcriptional sequencing described herein canaccommodate situations in which the rotation tag is from 10 nm to manymicrons from the surface.

FIG. 2 shows 2.7 micron magnetic beads before (FIG. 2A) and after (FIG.2B) application of a magnetic field.

Detection Methods

Detecting the rotational pattern of the rotation tag under varioussequencing conditions in the rotation-dependent transcriptionalsequencing methods described herein requires a light source, forprojecting light onto the rotation tag, and optics, for visualizing therotation tag and observing changes in the rotational pattern. While anynumber of suitable illumination methods and optics can be used in therotation-dependent transcriptional sequencing methods described herein,the following embodiments are provided as examples of the simplicity ofthe present systems and methods and the lack of any requirement forcomplex and expensive technologies for the detection component.

The light source can be LED, or the light source can be white light orsingle- or multi-fibers. The light source can be steady illumination orcan be provided as pulsed illumination, if desired. The light can beprojected from the same direction or from multiple directions. Forexample, the light source can be polymer fibers or bundles, which can betunable (e.g., in intensity and/or spectrum). Fresnel and/or Fraunhofferdiffraction can be used, based on the size of the rotation tag and itsdistance from the detector, to identify the rotation tag (e.g., theshape of the rotation tag) and identify its rotation.

The optics can be simple lenses and objectives (e.g., a microscope lenswith a 50× objective, e.g., Mitutoyo 50× with numerical aperture of0.75) and a tube lens with 1× magnification. The optics also can includea camera (e.g., a video camera, e.g., a scientific CMOS) operating atappropriate frames per second. Alternatively, the optics can utilizecurrent complementary metal-oxide-semiconductor (CMOS) or charge-coupleddevice (CCD) technology, provided they have an adequate number of pixels(and provided the light source provides sufficient photons to image therotation of the rotation tag). Currently used CMOS detectors are veryfast (e.g., 1000 or more frames per second, corresponding to exposuresof 1 ms or less). This level of detector speed removes any ambiguity dueto the stochastic nature of rapid nucleotide incorporation that plaguescurrent approaches. For example, many current approaches (e.g., PacificBiosciences, Complete Genomics, Inc.) are typically limited to exposuresof 10 ms or longer, due to the need to collect an adequate number offluorescent photons within the camera exposure window, which results inerrors in the sequence data collected.

The ability to use a small pixel size for the imaging detector isanother advantage of the present methods compared to other sequencingtechnologies (e.g., Pacific Biosciences, Complete Genomics Inc., andothers that rely upon single-molecule fluorescence or generalfluorescence at high throughput image capture). The rotation-dependenttranscriptional sequencing described herein can use a small pixel size(e.g., 1.4 microns, which is the current state of the art for cell phonecameras), compared to the 6.5 microns, which is the state of the art forthe CMOS sensors used in other real-time sequencing applications. Thisis primarily because the current sequencing methods can accommodate morenoise than other systems. Significantly, in the rotation-dependenttranscriptional sequencing described herein, the throughput of imagingcan be much higher because of the ability to use large sensors havingsmall pixels (e.g., 8-10 MPixels common in next-generation cell phones)and by imaging a larger area of the surface. The detection methods forthe rotation-dependent transcriptional sequencing methods describedherein allow for the use of commercial CMOS camera hardware with onlymodifications to the software, as opposed to other sequencing systems inthe industry, which rely upon specialized optical platforms (e.g.,ChemFet, Ion Torrent).

The data acquisition method in video mode from these sensors typicallyuses the intensity value of each pixel (e.g., using RAW format data).When the light source is a white LED or an RGB illuminator withappropriate balancing between the colors, diffraction and shadowpatterns of a rotation tag will be recorded in adjacent pixels and animage can be generated as if the sensor was a black and white sensor.For example, direct shadow or diffraction of, for example, anon-spherical rotation tag, can be recorded on the detector's pixels androtation can be detected due to the changing effects on diffraction.

Simply by way of example, detection of rotation (e.g., detection of thepattern of rotation by a rotation tag) can occur as follows. Rotationtags can be illuminated from above using, for example, an LED or fibercoupled—LED or LED array. Below the solid surface flow cell, a Mitutoyo50× objective having a numerical aperture of 0.75 that is infinitycorrected can be used in conjunction with a tube lens that images thefield of view onto a scientific CMOS chip (e.g., having 5.5 MP, with a6.5 micron center-to-center distance between pixels that areapproximately 6.5 micron size each). Assigning 10×10 pixels to eachrotation tag, such a pixel size is adequate to detect rotation of 1micron beads, which, on the entire CMOS surface, can result in more than55,000 rotation tags. If each rotation tag is attached to a 5 Kb targetnucleic acid molecule, the rotation-dependent transcriptional sequencingdescribed herein can sequence more than 275 Mbases in a single run on asingle chip. By way of comparison, the E. coli genome is 4.5 Mbases. Ifthe rate of transcription is 10 nucleotides per second, which is areasonable number for immobilized RNA polymerase, the complete E. coligenome could be sequenced as described herein in 500 seconds, i.e. lessthan 10 minutes. A detector that has the ability to record up to 100frames per second (i.e., 10 frames per each transcribed nucleotide) issufficient for the sequencing systems and methods described herein. Thecalculations above do not include any efficiency of attachment. Inaddition, in an asynchromous pattern of sequencing, assuming the pauseby RNA polymerase is less than 10 frames, it does not add any extrareaction time to the calculation above, however, the asynchronouspattern of sequencing could add time to the reaction if therate-limiting nucleoside triphosphate is tuned to much lower levels(e.g., to increase accuracy and minimize deletion errors).

Another example of detecting the rotational pattern of the rotation tagfollows. Rotation tags can be illuminated using an LED or fibercoupled—LED or LED array, where the rotation-dependent transcriptionalsequencing complexes are placed on top of a tapered waveguide having a350 nm core size on one side and a 6.5 micron core size on the other(about a 1:19 taper), which can be bonded to the surface of a scientificCMOS chip (e.g., Fairchild Imaging). Such a chip has 5.5 MPixels with6.5 micron center-to-center distance between pixels that are eachapproximately 6.5 microns. In this case, assigning 3×3 pixels to eachrotation tag, which is adequate to detect rotation of a 1 micron bead ontop of the 350 nm cores, allows for more than 610,000 rotation tags onsuch a chip. If each rotation tag is attached to a 5 Kb target nucleicacid molecule, a total of more than 3 Gbases can be sequenced in asingle run on a single chip. This is essentially single coverage of theentire human genome. The calculations above do not take into accountefficiencies of attachment, the rounding effects of the signal from theround cores of the taper to the square pixels, or the loss of pixels dueto mismatch in the bonding process of core-to-pixel.

The following is an example of detection of a rotational pattern in ahigh throughput sequencing system. For example, instead of using thetapered waveguide and scientific CMOS sensor described in the aboveexample, an OmniVision Inc. chip with, for example, 14.5 MP and 1.2microns per pixel, can be used after having removed the detector windowand attached one or more components of the rotation-dependenttranscriptional sequencing complexes directly on the microlens array ofthe chip. The rotation pattern using 2.8 micron rotation tags can bedetected on 2×2 pixels using signal processing of videos. Allowing foran extra 2 pixels in each direction for an array of rotation tags inproximity to each other, 8 pixels total can be assigned per rotationtag. In this case, a single chip can have more than 1.8 million rotationtags attached to target nucleic acid molecules being sequenced.Therefore, three of these chips would be adequate to sequence thecomplete human genome, while four chips could significantly increase theaccuracy. Electronics to produce a readout of this sensor in adequatespeeds (e.g., to match the nucleotide incorporation rate for thespecific chip) based on FPGA or other DSP designs and direct storage toarrays of solid state drives can be used and their components are knownin the art.

Simply by way of example, detection of rotation can occur as follows.LED structured illumination (e.g., Lightspeed genomics module adaptedfor speckle-free excitation with a 4× lens) can be used to create adiffractive shadow of a rotation tag on the surface of a SciCMOS camera(e.g., 5.5 MP at 100 frames per second). The interference pattern canexploit 5 frames per image in order to enhance resolution by 9×,effectively creating a 49 MP sensor at 20 fps. Each single molecule canbe assigned, for example, to a 5×5 pixel, which allows for about 2million rotation tags per chip. Assuming Poisson efficiencies at thetag-to-tag coupling, 660,000 available single RNA polymerase moleculeseach transcribing 5 Kb nucleic acid molecules results in 3.3 Gbases thatcan be sequenced in a single run. In the asynchronous sequencing method,four reactions can be performed (although, in some embodiments, threereactions are performed and the fourth nucleotide is inferred from thesequencing information obtained with the other three nucleosidetriphosphates). Allowing for 5 frames per enzyme pause with a total of250 ms at each pause, while the polymerase otherwise incorporates 15 ntper second, throughput is an extreme 18 Gb per hour. This sequencingrate is significantly higher than any current technologies, even forshort read lengths. As indicated herein, current cell phone cameratechnology (10 MP, 1.2 μm pixel size) meets the requirements of thissystem.

Similarly, color (e.g., with RGB filter coatings on the sensor) ormonochrome cell phone or digital camera sensors meet the requirementsfor use in the sequencing methods and systems described herein (e.g.,14.5 MP and a 1.25 micron pixel sensor). Even though each pixel of anRGB sensor is coated with a red or green or blue filter in a pattern,those sensors can be used in the sequencing systems and methodsdescribed herein since the light source used in the sequencing systemsand methods described herein can have a broad spectrum (e.g. whiteLED(s)) and provide scattered, reflected, diffracted or transmittedoptical signal to the detector portion of the camera chip (e.g.silicon).

In this example, the number of pixels used to detect each rotation tagcan be estimated by the shadow or diffraction of the rotation tag. Whendirect light illumination is used instead of fluorescence excitation,thick optical filters that block the excitation light and/or havefluorescence passbands are not necessary. These filters can be hundredsof microns thick, which can complicate the optical designs needed tocollect the fluorescence on a small number of pixels on the on-chipdetector. Collecting the light on a small number of pixels is importantsince one aspect of the throughput of sequencing systems is defined bythe total number of pixels on the detector divided by the number ofpixels assigned to each site or event. For example, with ani-Phone-style 8 MP CMOS sensor, assigning 10 pixels per site, allows fora total of 800,000 sites. If a 1 Kb nucleic acid, on average, issequenced per run per site, then, at most, throughput is 800 MBases perrun. Using Fraunhoffer diffraction and 3 micrometer beads at a distanceof 1 micrometer, then less than 10 pixels per site can be used withoutthe need for lenses having complicated optical designs.

In one embodiment, a source of tension can be provided that allows foran array of rotation tags to be elevated from the surface to an area,for example, greater than 4×4 mm². The present methods and systems arenot limited by the field of view (FOV), particularly when the sequencingis performed on-chip, where the effective FOV is the entire area of thechip. For example, the surface area of the Omnivision 14 MP sensor isabout 6×4 mm.

As indicated herein, one of the many advantages provided by therotation-dependent transcriptional sequencing methods described hereinis the extremely fast nucleotide incorporation by RNA polymerases due totheir stochastic nature. The high rate of incorporation and theresulting speed of rotation, however, can make data capture challenging.It would be understood by those skilled in the art that, in thepresently described asynchronous sequencing methods, for example, onlythe “end-point” information needs to be recorded, e.g., only the numberof total rotations until a pause occurs. That is, there is no need tocapture the individual incorporation steps, which may be very fast anddifficult for a simple detector to image effectively. Therefore, as longas a sequencing reaction is recorded at a high enough frame rate todetect the total amount of rotations between pauses by the RNApolymerase (due to the presence of a nucleoside triphosphate in arate-limiting amount), an accurate nucleic acid sequence can bedetermined.

In some embodiments, the rotational pattern of a magnetic rotation tagcan be captured using a GMR (giant magneto-resistance) array as thesensor. In some embodiments, a spin-valve array or MRAM can be used todetect the rotational pattern of a magnetic rotation tag. For example, acomputer memory array (e.g., RAM, e.g., DRAM) can be modified (e.g.,polished to remove layers from the surface that may be shielding themagnetic field effects) to accept a surface modification close to thememory cell capacitive elements. The induced electric field of amagnetic rotation tag can be sensed by the two-dimensional array ofcells/capacitors of the RAM. The cells of the RAM array then can be readdirectly using memory reading software following installation of thememory into a computer. In the case when MRAM is used as a sensor, flowcan be used to provide a source of tension to the target nucleic acidwithout the use of a magnetic field. Alternatively, a magnetic field canbe used as the source of tension in a field where the sensing signal ofthe MRAM cells is modified.

Sequencing Conditions

Referring again to FIG. 1A, the rotation-dependent transcription complex100 can be generated in a number of different fashions. For example,promoter-bound target nucleic acid molecules 10 can be provided to asolid surface 30 having RNA polymerase 20 immobilized thereon. In thisembodiment, the rotation tag 40 can be bound to the target nucleic acidmolecules 10 before or after the target nucleic acid molecules 10 arecomplexed with the immobilized RNA polymerase 20. In another example,the RNA polymerase 20 and the promoter-bound target nucleic acidmolecules 10 can be combined and then deposited on the solid surface 30.As indicated, the rotation tag 40 can be bound to the promoter-boundtarget nucleic acid molecules 10 before or after the complex isdeposited on the solid surface 30. In another example, rotation tags 40can be attached to the promoter-bound target nucleic acid molecules 10and contacted with RNA polymerase 20. The RNA polymerase 20 can beimmobilized on a solid substrate 30 before introducing the rotationtagged-target nucleic acid molecules 10, or the entire complex can bedeposited and immobilized on a solid substrate 30.

The rotation-dependent transcriptional sequencing described herein canbe performed in an asynchronous (i.e., rate-limiting) mode (FIG. 3A) ora synchronous (i.e., base-by-base) mode, or any combination thereof todetermine the sequence of a target nucleic acid molecule (FIG. 3B). At aminimum, “sequencing conditions,” as used herein, refers to the presenceof at least one nucleoside triphosphate, which can be used as describedbelow to determine the sequence of a target nucleic acid molecule.

In addition to the presence of at least one nucleoside triphosphate asdiscussed in more detail herein, conditions under which sequencingreactions are performed are well known in the art. For example,appropriate buffer components (e.g., KCl, Tris-HCl, MgCl₂, DTT,Tween-20, BSA) can be used to provide a suitable environment for theenzyme.

a) Asynchronous Sequencing

The rotation-dependent transcriptional sequencing method describedherein can be used to sequence nucleic acids based on an asynchronousincorporation of nucleotides. For asynchronous embodiments, thesequencing conditions under which the initial reaction occurs (i.e.,first sequencing conditions) include the presence of four nucleosidetriphosphates, where the nucleoside triphosphates are present indifferent amounts, at least one of which is rate-limiting and at leastone of which is not rate-limiting. For example, one of the fournucleoside triphosphates is provided in a rate-limiting amount (e.g., inan amount that is less than the amount of the other three nucleosidetriphosphates). In such a reaction, the RNA polymerase will effectivelypause each time it tries to incorporate the nucleoside triphosphateprovided in the rate-limiting amount into the transcript, and such apause can be observed in the rotational pattern of the rotation tag asdescribed herein.

Significantly, the number of bases between each pause can be preciselydetermined by detecting the cumulative amount of rotation betweenpauses. Thus, the precise position of, for example, each guanine (G)nucleotide along the sequence of the target nucleic acid molecule can beconcisely determined due to changes in the rotational pattern when the Gnucleoside triphosphate is provided in rate-limiting amounts. Similarreactions can be performed under second, third and, if desired, fourth,sequencing conditions in which, respectively, the second, third, andfourth nucleoside triphosphate of the four nucleoside triphosphates ispresent in a rate-limiting amount. As shown in FIG. 4, the combinedinformation from the four reactions, whether they are performedsimultaneously with one another or sequentially following one another,provide the complete sequence of the target nucleic acid molecule.

The pattern, even from a single reaction resulting in the positionalsequence of one of four nucleotides can be compared to nucleic aciddatabases and used to identify the nucleic acid molecule with a highlevel of confidence. In addition, it would be understood by thoseskilled in the art that the sequence of a target nucleic acid moleculecould be compiled using the positional information produced from threeof the four nucleoside triphosphates, as the positional information ofthe fourth nucleotide in the sequence can be inferred once the otherthree nucleotides are known.

b) Synchronous or Base-By-Base Sequencing

The rotation-dependent transcriptional sequencing method describedherein can be used to sequence nucleic acids in a synchronous pattern,which otherwise might be known as base-by-base sequencing. Forsynchronous or base-by-base embodiments, the sequencing conditions underwhich the initial reaction occurs (i.e., first sequencing conditions)include the presence of a single nucleoside triphosphate. In such areaction, transcription by the RNA polymerase will only proceed if thetarget nucleic acid contains the complementary base at that position,which can be observed as a change in the rotational pattern of therotation tag as described herein. Based on the structuralcharacteristics of the double-helix, incorporation of one nucleotide byRNA polymerase results in about a 36 rotation. Such reaction conditionsare continued until the rotational pattern of the rotation tag does notchange. It would be understood that the cumulative change in therotational pattern can be used to precisely determine the number oftimes the first nucleoside triphosphate was sequentially incorporatedinto the transcript (e.g., in a homopolymeric region of the targetnucleic acid molecule).

When a change is no longer observed in the rotational pattern of therotation tag under the first sequencing conditions (i.e., the presenceof a first nucleoside triphosphate of the four nucleosidetriphosphates), or if no changes in the rotational pattern are observedunder the first sequencing conditions, a reaction is performed undersecond sequencing conditions. Second sequencing conditions include thepresence of a second nucleoside triphosphate of the four nucleosidetriphosphates. Changes in the rotational pattern of the rotation tag areindicative of transcription (i.e., the incorporation of one or more ofthe particular nucleoside triphosphate present into the transcript bythe RNA polymerase), while the absence of a change in the rotationalpattern of the rotation tag indicates that no transcription took place.

Such reactions, under first sequencing conditions, second sequencingconditions, third sequencing conditions (i.e., the presence of a thirdnucleoside triphosphate of the four nucleoside triphosphates) or fourthsequencing conditions (i.e., the presence of a fourth nucleosidetriphosphate of the four nucleoside triphosphates), can be carried outin such a manner that the sequence of the target nucleic acid moleculeis sequentially determined based on the changes in the rotationalpattern and/or cumulative angle of rotation of the rotation tag undereach of the respective sequencing conditions. It would be understood bythose skilled in the art that steps can be taken to remove the residualnucleoside triphosphates under one sequencing condition beforeintroducing a different sequencing condition. For example, the surfaceon which the RNA polymerase is immobilized can be washed or flushedbefore introducing a different nucleoside triphosphate. While suchwashing steps are not required, it would be understood that such stepswould increase the accuracy of the resulting sequence information.

c) Additional Sequencing Methodologies

The rotation-dependent transcriptional sequencing methods describedherein are amenable to a number of different variations and routinemodifications, which can be utilized, for example, and withoutlimitation, to increase the accuracy of the sequencing information andincrease the amount of information obtained in a sequencing reaction.

For example, many RNA polymerases possess a “strand-switching” or“turn-around transcription” ability. This feature can be advantageouslyused in the methods described herein to increase the accuracy of theresulting sequence information. For example, when RNA polymerase reachesthe end of a target nucleic acid, the RNA polymerase can “jump” to theopposite strand and continue transcription. See, for example, McAllisterat al. (US 2007/0077575) and Rong et al. (1998, “Template StrandSwitching by T7 RNA Polymerase”, J. Biol. Chem., 273(17):10253-60). Inaddition, certain RNA polymerases can “jump” from the double-strandedDNA template to the hybrid DNA-RNA transcript and resume transcriptionof the DNA strand. In addition, this type of recursive sequencing of atarget nucleic acid molecule can be genetically engineered byintroducing (e.g., ligating) a RNA polymerase promoter onto each end ofthe target nucleic acid molecule, such that the RNA polymerase binds andtranscribes the opposite strand.

In addition, one or more different RNA polymerases (e.g., RNApolymerases from different organisms or different RNA polymerases fromthe same organism) can be immobilized onto a solid surface. As is knownin the art, different RNA polymerases recognize and bind to differentpromoter sequences. Therefore, one or more different RNA polymerasepromoters can be ligated to different populations of target nucleic acidmolecules and a combined population of target nucleic acid molecules canbe sequenced, based on the rotational pattern of the rotation tag, usingthe one or more different RNA polymerases immobilized on the solidsurface. By differentially-labeling, for example, the different RNApolymerases or the different populations of target nucleic acidmolecules (using, for example, beads emitting different wavelengths,fluorescent tags, or fluorescently-labeled antibodies), the sequence ofone population of target nucleic acid molecules can be distinguishedfrom the sequence of another population of target nucleic acidmolecules. Using such methods, sequencing reactions on differentpopulations of target nucleic acid molecules can take placesimultaneously.

In some embodiments, both the RNA polymerases and the populations oftarget nucleic acid molecules can be differentially labeled. It would beunderstood that labeling the target nucleic acid molecules can occurdirectly via the nucleic acid or, for example, via the rotation tag.This ability to differentially label at multiple levels of thesequencing reaction can be used, for example, to compare theprocessivity of different RNA polymerases on target nucleic acidmolecule having the same sequence, which may identify, for example,homopolymeric regions or regions of methylation, or to compare thetranscription of target nucleic acid molecules having differentsequences by more than one RNA polymerase.

Simply by way of example, any combination of RNA polymerase enzymes(e.g., from one or more of the T7, T3, SP6 or K11 bacteriophages), inconjunction with the appropriate nucleic acid promoter sequences, can beused in the rotation-dependent transcriptional sequencing methodsdescribed herein. As discussed herein, this feature allows for amultiplexing of the sequencing reactions. Other variations that utilizedifferent RNA polymerases in conjunction with their specific promotersequences as well as differential-labeling techniques are contemplatedherein.

In some embodiments, two asynchronous rotation-dependent transcriptionalsequencing reactions can be performed under the same sequencingconditions (e.g., first sequencing conditions). Once sequencing hasprogressed for a sufficient number of nucleotides (e.g., at least 100nt, 500 nt, 1,000 nt, 5,000 nt, or 10,000 nt), the sequencing conditionsof one of the reactions can be changed (e.g., to second sequencingconditions), and the rotation-dependent transcriptional sequencingcontinued. The resulting sequence information obtained under the firstsequencing conditions can be used to align a particular target nucleicacid molecule in the first reaction with the same particular targetnucleic acid molecule in the second reaction, which, when the sequencingconditions are changed, allows positional sequence information to beobtained for two nucleotides within a particular target nucleic acidmolecule.

Those skilled in the art would understand that, due to the torsion placeon the nucleic acid molecules by the RNA polymerase, the rotation tagmay produce a “load”, possibly slowing down the RNA polymerase. This canbe prevented or diminished, for example, by using a rotation tag havinga different shape (e.g., two oblong beads or a bead-rod combination),which can result in more friction in the fluidic medium than a simplespherical bead. On the other hand, there may be RNA polymerases and/orsequencing conditions in which a mechanical loading of the RNApolymerase can be used to advantageously affect the rate of sequencing.

Articles of Manufacture/Kits

Articles of manufacture (e.g., kits) are provided herein. An article ofmanufacture can include a solid substrate, as discussed herein, ontowhich a plurality of RNA polymerase enzymes is immobilized. A pluralityof RNA polymerase enzymes refers to at least 10 RNA polymerases (e.g.,at least 20, 50, 75, or 100 enzymes), at least 100 RNA polymerases(e.g., at least 200, 500, or 1,000 enzymes), or at least 1,000 RNApolymerases (e.g., at least about 2,500, 5,000, 10,000, or 50,000enzymes).

Articles of manufacture are well known in the art and can includepackaging material (e.g., blister packs, bottles, tubes, vials, orcontainers) and, in addition to the solid surface having RNA polymerasesimmobilized thereon, can include one or more additional components.

In some embodiments, an article of manufacture can include a rotationtag. As described herein, the rotation tag can be a non-spherical tag ora spherical tag having a non-uniform feature that can be used to detectrotation.

In some embodiments, an article of manufacture can include nucleic acidsequences corresponding to a RNA polymerase promoter. As discussedherein, promoters that direct transcription by RNA polymerases are wellknown and used routinely in the art.

In some embodiments, an article of manufacture can include a tether. Asdiscussed herein, a tether can be used to attach target nucleic acidmolecules to rotation tags. In some embodiments, a tether includesnucleic acid sequences, which, for example, can be biotinylated, suchthat they bind to streptavidin-labeled beads.

In some embodiments, an article of manufacture can include one or morenucleoside triphosphates. When more than one nucleoside triphosphate isprovided, they can be provided in combination (e.g., in a singlecontainer) or separately (e.g., in separate containers).

In some embodiments, an article of manufacture further includesinstructions. The instructions can be provided in paper form or in anynumber of electronic forms (e.g., an electronic file on, for example, aCD or a flash drive, or directions to a site on the internet (e.g., alink). Such instructions can be used to identify rotation of therotational tag relative to an axis through the magnetic reference tag,compile the sequence of a target nucleic acid molecule based on therotational pattern and the presence of a nucleoside triphosphate; and/orapply an appropriate tension on the nucleic acid.

Rotation-Dependent Transcriptional Sequencing Systems

A rotation-dependent transcriptional sequencing system as describedherein includes at least a Sequencing Module. A Sequencing Module forsequencing target nucleic acid molecules typically includes a receptaclefor receiving a solid substrate, a tension source for providingdirectional force, a light source for projecting light onto a rotationtag, and optics for detecting the pattern of rotation of the rotationtag. The tension source, light source, and optics are discussed herein.A receptacle for receiving a solid substrate can be configured, forexample, as a recessed chamber. Generally, a solid substrate for use ina rotation-dependent transcriptional sequencing system will have aplurality of RNA polymerases immobilized thereon, and, for a highthroughput sequencing system, the solid surface can be an on-chipembodiment as described herein. A Sequencing Module also can include acomputer processor or means to interface with a computer processor.Further, primary analysis software can be provided as part of aSequencing Module.

In addition, a Sequencing Module further can include a heating andcooling element and a temperature control system for changing andregulating the temperature of the sequencing reactions. In addition, aSequencing Module further can include fluidics (e.g., one or morereagent or buffer reservoirs and tubing for delivering the one or morereagents or buffers to the reaction chamber (e.g., the chip)). Fluidicsfor delivering one or more reagents or buffers also can include, withoutlimitation, at least one pump. Without limitation, exemplary reagentsthat can be used in a sequencing reaction can include, for example,nucleoside triphosphates, enzymes (RNA polymerase) and rotation tags.Also without limitation, exemplary buffers that can be used in asequencing reaction can include, for example, of a wash buffer, anenzyme-binding buffer and a sequencing buffer.

FIG. 5 is a schematic showing a representative flow cell 70. In the flowcell 70 shown, a laser-cut Pressure Sensitive Adhesive (PSA) filmapproximately 50 microns thick is laid on top of an OmniVision sensor(OV14810; after removal and coating of the window with SiN). Thisconfiguration results in a flow cell on top of a CMOS chip 80. A glassor polymeric slide, having holes for microfluidic feed-through tubes 90,is then applied. Multiple rotation-dependent transcriptional complexes100 are shown in the field. FIG. 6 shows a photograph (Panel A) and aline drawing (Panel B) of a representative flow cell 70 in which fibers60 are used to deliver light and a microscope objective 200 is used forimaging. The solid surface 30 also is shown.

FIG. 7 is a schematic showing a rotation-dependent transcriptionalsequencing system 300. The schematic shows the tension source 50, thelight sources 60, and the flow cells 70 with in- and out-fluidics 90.The flow cells 70 in FIG. 7 are shown on a chip 80 (e.g., CMOS, CCD, ormodified versions thereof).

FIGS. 8A and 9A show photographs of different embodiments of arotation-dependent transcriptional sequencing system 300 as describedherein, and FIGS. 8B and 9B are the respective line drawings of theembodiments shown in each photograph. Referring to FIGS. 8B and 9B, bothof the rotation-dependent transcriptional sequencing systems 300 show asolid surface 30, a light source 60, and fluidics 90 for delivering andremoving the various reagents and/or buffers. A tension source 50 isshown in each of FIGS. 8 and 9; in FIG. 9, the tension source 50 ismoved out of the way to more easily access the flow cell on the solidsurface 30. The rotation-dependent transcriptional sequencing systems300 in FIGS. 8 and 9 both utilize similar optics 200. The system 300 inFIG. 8 utilizes a 20× water-objective 200 (here, a “super-lens” fromOlympus, which provides more than 1 mm FOV with NA=0.9, i.e. highresolution with a large field of view), and the system 300 in FIG. 9utilizes a 20× objective tube lens 200. Both FIGS. 8 and 9 also includea camera 200.

In one embodiment, structured illumination and stroboscopy can be usedin a rotation-dependent transcriptional sequencing system as follows.For example, two or more fibers each can be coupled to a separate LEDand their illumination directed in different angles with respect to theplane of the solid surface. The light sources can be independentlycontrolled in light intensity (or “amplitude”), in spectral content(e.g. different color LEDs or filtered white LEDs) and in time (e.g. viatriggering the electronics from a single trigger but after differentdelays incorporated in the timing of the trigger). The same trigger,but, for example, at a different delay or amplitude or shape, caninitiate the exposure of the camera. Since the exposure duration of thecamera can be controlled separately, it is feasible to time the firstlight source (e.g., reflection can be captured, for example, by the evennumber frames) and the second light source (e.g., reflection can becaptured, for example, by the odd number frames) to illuminate therotation tag and its signature. Since the illumination is at differentangles (e.g. one from the top and one from the bottom, i.e., in anepiposition, or one from top and one at, e.g., a 45° angle to thevertical), the rotation tag can be detected in one image and not in theanother image. Since a series of images is captured, and since more thanone source can be used, it is possible to reconstruct virtually athree-dimensional shape of the rotation tag in every frame. This levelof detection leads to higher accuracy and fewer deletion errors in thefinal sequence.

The rotation-dependent transcriptional sequencing systems describedherein can significantly advance point-of-care diagnostics and genomicsbased on massively parallel single molecule analysis with the singlenucleotide resolution. The system is intrinsically suited for highlymultiplexed target identification and has unlimited flexibility in beingable to be reconfigured to interrogate simultaneously or sequentiallydifferent nucleic acid targets, e.g. pathogens and human biomarkers. Inaddition, while current PCR- and microarray-based methods of sequencingnucleic acids are limited by being able to detect only known sequencesor infectious agent(s) because of the specific set of reagents (primersand probes) required for positive identification.

For a system designed, for example, for high-throughput clinicaldiagnostics or for point-of care diagnostics, a rotation-dependenttranscriptional sequencing system as described herein can be coupledwith a Sample Preparation Module and a Template Finishing Module.

A Sample Preparation Module can be configured to lyse cells, therebyreleasing the nucleic acids, and a Sample Preparation Module also canhave the capability of shearing/fragmenting the nucleic acid. A SamplePreparation Module typically includes a receptacle for receiving abiological sample, and fluidics for delivering one or more reagents orbuffers to the biological sample. A Sample Preparation Module can beconfigured to receive a variety of different biological samples or aSample Preparation Module can be configured to receive a specific typeof biological sample (e.g., a swab, a tissue sample, a blood or plasmasample, saliva, or a portion of a culture) or a biological sampleprovided in a specific form (e.g., in a vial or tube or on blottingpaper). A Sequencing Preparation Module also can be configured tocapture certain molecules from the biological sample (e.g., bacterialcells, viruses, etc.) using, for example, filters, columns, magnets,immunological methods, or combinations thereof (e.g., Pathogen CaptureSystem, NanoMR Inc.).

A Sample Preparation Module can include reagents or buffers involved inobtaining the nucleic acids from a biological sample and preparing thenucleic acids for sequencing. For example, reagents involved inobtaining nucleic acids for sequencing include cell lysis reagents,nucleic acid cleavage enzymes, DNA polymerases, oligonucleotides, and/orDNA binding agents (e.g., beads or solid matrices to bind and wash thetarget nucleic acid molecules), while buffers involved in obtainingnucleic acids for sequencing include lysis buffer, wash buffer, elutionbuffer, or binding buffer. Since the rotation-dependent transcriptionalsequencing described herein requires double-stranded nucleic acidtemplates, a Sample Preparation Module can include the necessaryreagents and buffers to convert single-stranded DNA or RNA todouble-stranded nucleic acid molecules (e.g., PCR reagents).

Many of the functional components of a Sample Preparation Module arecommercially available (e.g. Silica gel membrane (Qiagen or Ambion kits)or as an integrated part of Palladium System (Integrated NanoTechnologies Inc.)). In addition, as an alternative to enzymaticcleavage of nucleic acid templates, instruments that fragment nucleicacids are commercially available (e.g., Covaris).

A Template Finishing Module can be configured to attach RNA polymerasepromoter sequences and rotation tags to target nucleic acid molecules. ATemplate Finishing Module typically includes fluidics for delivering oneor more reagents or buffers to the target nucleic acid molecules. Forexample, a Template Finishing Module can include reagents and buffersfor the purpose of ligating RNA polymerase promoter sequences to thetarget nucleic acid molecules, and a Template Finishing Module also caninclude reagents and buffers for attaching a rotation tag to the targetnucleic acid molecules. For example, reagents involved in ligatingpromoter sequences or binding rotation tags to target nucleic acidmolecules include, obviously, the promoter sequences and the rotationtags, but also can include, for example, ligase enzymes, a tether or PCRreagents, while buffers involved in ligating promoter sequences orbinding rotation tags to target nucleic acid molecules include ligationbuffer, rotation tag-binding buffer, enzyme-binding buffer, washingbuffer and sequencing buffer.

Depending upon the configuration of the rotation-dependenttranscriptional sequencing system as described herein, the plurality ofRNA polymerases can be immobilized on the solid surface prior tointroducing the promoter- and rotation tag-bound target nucleic acidmolecules. Alternatively, the plurality of RNA polymerases can becombined with the promoter- and rotation tag-bound target nucleic acidmolecules and the entire complex deposited on the solid surface. Thelatter procedure is feasible because the binding kinetics for RNApolymerases and their corresponding promoter sequences is very fast,efficient and specific.

Sequence Determination Following Rotation-Dependent TranscriptionalSequencing

FIG. 10 shows a screenshot of video and the corresponding digitizationof the rotational pattern of a rotation tag (top), which is then shownin graphical form (bottom). The rotation tag is a 2.8 micronstreptavidin-coated bead with a 1 micron biotin-coated bead attachedthereto. Transcription was performed by a His-tagged RNA polymerase inthe presence of all four nucleoside triphosphates. FIG. 10 demonstratesthat a rotational pattern of a rotation tag can be clearly and reliablydetected and measured from the images captured with a detector.

FIG. 11A is a flow diagram illustrating rotational analysis. A number ofmetrics can be determined from the rotational analysis, including atimestamp, displacement (from n=1 and n−1), displacement boundingcircle, bead diameter, doublet orientation, accumulated rotation,angular velocity, and ellipse eccentricity. Image segmentation can beused to separate each pixel into two classes, foreground (the rotationtag) and the background. Using one method (the Otsu's method), the imagehistogram and the probabilities of each intensity level can becalculated, and then the threshold that minimizes intra-class variancecan be determined. Using another method (Triangle method), a line isdrawn between the max of the histogram at b on the gray level axis andthe lowest value a on the gray level axis. The distance L normal to theline and between the line and the histogram is computed for all valudesfrom a to b. The level where the distance between the histogram and theline is maximal corresponds to the threshold value. Using yet anothermethod (Adaptive mean or median), a localized window (i.e., 5×5 pixels)can be used to find the maximum intensity variance, which then is usedto compute the mean or median for that window.

The bounding ellipse method can utilize any of several methods: thecentroid method, which is normalized to the 2^(nd)-order moments of theforeground (rotation tag) region; the ellipse major axis method, which,on the centroid of the region, the maximum distance to the region edgeis the first point of the major axis and the reflection (from thecentroid) is the 2^(nd) point; the ellipse minor axis method, which, onthe centroid of the region, is the maximum distance to the region edgeand perpendicular to the major axis; and the ellipse orientation method,which is the angle between the ellipse major axis and the x axis.

FIG. 11B is a flow diagram illustrating an example process 1100 fordetermining the sequence of a target nucleic acid molecule. In someexamples, the process 1100 can be implemented using one or more computerprogram applications executed using one or more computing devices. Forpurposes of illustration, a non-limiting example context is providedthat is directed to determining the sequence of a target nucleic acidmolecule comprising a rotation tag based upon data obtained duringtranscription of the target nucleic acid molecule.

The process 1100 starts by setting an identified position to the currentnucleic position in a target nucleic acid molecule (1110) beingsequenced using the rotation-dependent transcriptional sequencingdescribed herein. An identified position can be, for example, the firstnucleotide transcribed, the first nucleotide transcribed from the targetnucleic acid molecule (i.e., after the promoter sequences), or anynucleotide position along a target nucleic acid molecule.

First datum (i.e., first information) at the identified position in thetarget nucleic acid molecule is received (1120) from therotation-dependent transcriptional sequencing system or provided basedupon information from the operation of the rotation-dependenttranscriptional sequencing, and second information (i.e., second datum)at the identified position in the target nucleic acid molecule isprovided or received (1120). For example, the first datum can beinformation regarding rotation of the rotation tag. For example, firstdatum can be a rate of rotation (i.e., degrees of rotation/time), adetermination of the presence or absence of rotation, or a change in anestablished rotational pattern. For example, the second datum can beinformation regarding the presence and/or availability (e.g.,concentration) of one or more nucleoside triphosphates in the sequencingreaction.

The nucleotide at an identified position then can be determined basedupon the first and second data. For example, if the first datumindicates a change in the rotational pattern and the second datumindicates the presence of guanine nucleoside triphosphate in thereaction, then the nucleotide at the identified position in the targetnucleic acid molecule is determined to be cytosine. Similarly, if thefirst datum indicates an absence of change in the rotational pattern andthe second datum indicates the presence of guanine nucleosidetriphosphate in the reaction, the nucleotide at the indicated positionin the target nucleic acid molecule is determined to be non-guanine(i.e., adenine, guanine, and thymine).

If it is determined that the identified position can be advanced to anext position (1140), the identified position is set equal to the nextnucleic position in the target nucleic acid molecule (1150) and theprocess 1100 continues (1120). If it is determined that the identifiedposition cannot be advanced to a next position (1140), the sequence ofthe target nucleic acid molecule based on the first information andsecond information received at each identified position is compiled(1160) and the process 1100 ends. The identified position cannot beadvanced to a next position when transcription can no longer occur due,for example, to completion of transcription of the target nucleic acidmolecule or expiration of RNA polymerase activity (e.g., due to decay ofenzyme activity).

Embodiments of the subject matter and the operations described in thisspecification can be implemented in digital electronic circuitry, or incomputer software, firmware, or hardware, or in combinations of one ormore of them. Embodiments of the subject matter described herein can beimplemented as one or more computer programs, i.e., one or more modulesof computer program instructions, encoded on computer storage medium forexecution by, or to control the operation of, data processing apparatus.Alternatively or in addition, the program instructions can be encoded onan artificially generated propagated signal, e.g., a machine-generatedelectrical, optical, or electromagnetic signal that is generated toencode information for transmission to suitable receiver apparatus forexecution by a data processing apparatus. A computer storage medium canbe, or be included in, a computer-readable storage device, acomputer-readable storage substrate, a random or serial access memoryarray or device, a mobile communication device, or a combination of oneor more of them. Moreover, while a computer storage medium is not apropagated signal, a computer storage medium can be a source ordestination of computer program instructions encoded in an artificiallygenerated propagated signal. The computer storage medium can also be, orbe included in, one or more separate physical components or media (e.g.,multiple CDs, disks, or other storage devices).

The operations described herein can be implemented as operationsperformed by a data processing apparatus on data stored on one or morecomputer-readable storage devices or received from other sources. Theterm “data processing apparatus” encompasses all kinds of apparatus,devices, and machines for processing data including, by way of example,a programmable processor, a mobile communications device, a computer, asystem on a chip, or multiple ones, or combinations, of the foregoing.The apparatus can include special purpose logic circuitry, e.g., an FPGA(field programmable gate array) or an ASIC (application specificintegrated circuit). The apparatus can also include, in addition tohardware, code that creates an execution environment for the computerprogram in question, e.g., code that constitutes processor firmware, aprotocol stack, a database management system, an operating system, across-platform runtime environment, a virtual machine, or a combinationof one or more of them. The apparatus and execution environment canrealize various different computing model infrastructures, such as webservices, distributed computing and grid computing infrastructures.

A computer program (also known as a program, software, softwareapplication, script, or code) can be written in any form of programminglanguage, including compiled or interpreted languages, declarative orprocedural languages, and it can be deployed in any form, including as astandalone program or as a module, component, subroutine, object, orother unit suitable for use in a computing environment. A computerprogram may, but need not, correspond to a file in a file system. Aprogram can be stored in a portion of a file that holds other programsor data (e.g., one or more scripts stored in a markup languagedocument), in a single file dedicated to the program in question, or inmultiple coordinated files (e.g., files that store one or more modules,sub programs, or portions of code). A computer program can be deployedto be executed on one computer or on multiple computers that are locatedat one site or distributed across multiple sites and interconnected by acommunication network.

The processes and logic flows described herein can be performed by oneor more programmable processors executing one or more computer programsto perform actions by operating on input data and generating output. Theprocesses and logic flows can also be performed by, and apparatus canalso be implemented as, special purpose logic circuitry, e.g., an FPGAor an ASIC.

Processors suitable for the execution of a computer program include, byway of example, both general and special purpose microprocessors, andany one or more processors of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read only memory ora random access memory or both. The essential elements of a computer area processor for performing actions in accordance with instructions andone or more memory devices for storing instructions and data. Generally,a computer will also include, or be operatively coupled to receive datafrom or transfer data to, or both, one or more mass storage devices forstoring data, e.g., magnetic, magneto optical disks, or optical disks.However, a computer need not have such devices. Moreover, a computer canbe embedded in another device, e.g., a mobile communications device, apersonal digital assistant (PDA), a mobile audio or video player, a gameconsole, a Global Positioning System (GPS) receiver, or a portablestorage device (e.g., a universal serial bus (USB) flash drive), to namejust a few. Devices suitable for storing computer program instructionsand data include all forms of non volatile memory, media and memorydevices, including by way of example semiconductor memory devices, e.g.,EPROM, EEPROM, and flash memory devices; magnetic disks, e.g., internalhard disks or removable disks; magneto optical disks; and CD ROM andDVD-ROM disks. The processor and the memory can be supplemented by, orincorporated in, special purpose logic circuitry.

To provide for interaction with a user, embodiments of the subjectmatter described in this specification can be implemented on a computerhaving a display device, e.g., a CRT (cathode ray tube) or LCD (liquidcrystal display) monitor, for displaying information to the user and akeyboard and a pointing device, e.g., a mouse or a trackball, by whichthe user can provide input to the computer. In addition, a computer caninteract with a user by sending documents to and receiving documentsfrom a device that is used by the user; for example, by sending webpages to a web browser on a user's client device in response to requestsreceived from the web browser.

Embodiments of the subject matter described in this specification can beimplemented in a computing system that includes a back end component,e.g., as a data server, or that includes a middleware component, e.g.,an application server, or that includes a front end component, e.g., aclient computer having a graphical user interface or a Web browserthrough which a user can interact with an implementation of the subjectmatter described in this specification, or any combination of one ormore such back end, middleware, or front end components. The componentsof the system can be interconnected by any form or medium of digitaldata communication, e.g., a communication network. Examples ofcommunication networks include a local area network (“LAN”) and a widearea network (“WAN”), an inter-network (e.g., the Internet), andpeer-to-peer networks (e.g., ad hoc peer-to-peer networks).

The computing system can include clients and servers. A client andserver are generally remote from each other and typically interactthrough a communication network. The relationship of client and serverarises by virtue of computer programs running on the respectivecomputers and having a client-server relationship to each other. In someembodiments, a server transmits data (e.g., an HTML page) to a clientdevice (e.g., for purposes of displaying data to and receiving userinput from a user interacting with the client device). Data generated atthe client device (e.g., a result of the user interaction) can bereceived from the client device at the server.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Solid Surface Preparation

An NTA monolayer was prepared as described (see Paik et al., 2005, Chem.Commun., 15:1956-58. Ni-NTA surfaces were obtained by immersing theNTA-functionalized substrates into 10 mM Tris-HCl buffer (pH 8.0)containing 0.1 M NiCl₂ for 30 min. The substrates were then rinsedseveral times with Milli-Q water and dried under a nitrogen stream.

The freshly cleaned substrates were immersed into a distilled toluenesolution containing 1% (v/v) 3-glycidyloxypropyl trimethoxysilane underargon for 2 days. After the substrates were removed from the solution,they were rinsed with distilled toluene and dried under a nitrogenstream. The substrates functionalized with epoxy-terminated SAM wereincubated in 10 mM Tris-HCl buffer (pH 8.0) containing 2.5 mM N,Nbis(carboxymethyl)-L-lysine (NTA) at 60° C. for 4 h. The substrates wererinsed with Milli-Q water and dried in preparation for microcontactprinting.

A limited nonspecific binding effect of His-tagged protein to the NTASAM was observed, demonstrating the NTA SAM to be a suitable surface forfabricating Ni(II) ion patterns with microcontact printing and dip-pennanolithography techniques.

Example 2 Cloning and Purification of His-Tagged RNA Polymerase

A DNA fragment that encodes the 38 amino acid SBP-tag was synthesized byPCR using pTAGk19 as a template and synthetic DNA oligomers RP46 andRP47 (see below) as primers. The fragment was digested with NcoI andligated into pBH16117, resulting in pRP6.

SBP-His-RNA polymerase and His-RNA polymerase were expressed andpurified as previously described (He et al., 1997, J. Protein ExpressionPurif., 9:142-51; and Keefe et al, 2001, J. Protein Expression Purif.,23:440-46).

Example 3 Immobilization of RNA Polymerase

The following reaction scheme was followed for the immobilization of RNApolymerase molecules on Si(111): (a) 40% NH₄F, 10 min, 25° C.; (b) Cl₂gas, 20 min, 100° C.; (c) mPEG, over-night, vacuum, 150° C.; (d) DSC,DEIDA, DMAP, DMF, overnight, 25° C.; (f) BBTO, diethyl ether, 6 h, 25°C.; (g) CuSO₄, ethanol 20 min, 25° C.; (h) 6×His-tagged proteinincubation.

Example 4 Microcontact Printing (μCP) and Complex Formation

A 10:1 (v/v) mixture of poly(dimethylsiloxane) (PDMS) and curing agent(Sylgard 184, Dow Corning) was cast against a patterned silicon masterto prepare PDMS stamps with 5 micron line features, with a spacing of 3and 10 micron line features and a spacing of 5 micron. The non-oxidizedPDMS stamps were incubated in 10 mM Tris-HCl buffer (pH 8.0) containing0.1 M NiCl₂ for about 1 h and then dried with a nitrogen stream. Thestamps were brought into contact with a NTA-terminated substrate for 3min. After peeling off the stamp, the Ni(II)-printed substrates wereincubated in about 200 μL of 25 mM Tris-HCl buffer (pH 7.5) containing100 nM of His-T7 RNAP with ds-DNA, promoter and magnetic tags attachedvia streptavidin-biotin bonds for 30 min and then rinsed with 10 mMTris-HCl buffer (pH 8.0) and Milli-Q water to remove excess protein.

Example 5 Tethering and Rotation

2.8 micron SA-conjugated beads (Dynal) and 1.0 micron biotinylated beadswere diluted (1:20 and 1:200, respectively) in PBS, and mixed at roomtemperature for 15 min. Coverslips were coated with Ni2+-NTA HRPconjugate (Qiagen) and flow chambers were assembled by aligning togetherslightly separated coverslips as previously described (see, Noji et al.,1997, Nature, 386:299-302).

A 4 kb DNA template biotinylated at one end was mixed with SA beaddoublets and incubated with 20 nM His-T7 RNAP, 0.3 mM GTP, and 0.1 mMATP for 2 min to allow the formation of an elongation complex. Thesample (˜30 μl) was injected into a flow cell, incubated for 5 min, anda magnetic force of 0.1 pN was applied. The flow cells were washed withTB followed by addition of 0.5 mM NTPs.

It was found that positioning the magnet offset from the bead location,i.e. not directly on top but creating an angle with respect to the lightsource and objective, allows for easier calibration of positionalchanges and forces.

Example 6 Formation of Rotation-Dependent Transcriptional SequencingComplex

FIG. 2C shows an array of rotation tags tethered by a His-tagged RNApolymerase and a 5.1 Kb DNA template on a passivated, nickel-coatedglass slide. The “semirandom” array was created via magnetic fieldapplication that allows the beads to position themselves in ordereddistance with respect to each other. Singlet-, doublet- andtriplet-bead-carrying nucleic acids were attached to the surface byflipping the slide upside down so that a conical magnetic field pulledthe magnetic tags onto the glass surface in an ordered fashion.Percolation of magnetic beads in two-dimensional space actively createdthe pattern shown in FIG. 2C. Subsequently, the magnet was removed andthe flow chamber was flipped upside down again for the magnet to applytension to the nucleic acids. The sequencing reaction mix was thendelivered to the flow chamber to initiate transcription and sequencingof all molecules in parallel.

Example 7 Template Preparation

DNA template for Sequencing by transcription was prepared by joiningtogether 4.6 kb phage T7 DNA fragment bearing T7 promoter and 0.5 kbbiotinylated fragment of Lambda DNA. A 4.6 kb fragment was generated byPCR using #T7pPK13 forward primer and # T7phi17REV primer containing anXbaI recognition site at the 3′ end. A 0.5 kb PCR fragment was generatedby PCR using #F3 and #R3 primers in the presence of Biotin-16-dUTP(Roche). After PCR was completed, the purified PCR product was digestedwith NheI and cleaned up with QIAquick PCR Purification Kit (Qiagen).

After digestion of the PCR product with XbaI, the 4.6 kb piece wasjoined by overnight ligation at 15° C. with a 0.5 kb biotinylated PCRfragment digested with NheI. The resulting ligation product of 5.1 kbwas resolved using 0.7% agarose gel electrophoresis and extracted fromthe gel using QIAquick Gel Extraction Kit (Qiagen). This DNA was used inthe transcription and sequencing experiments.

The following primers were used for PCR: # T7pPK13: GCA GTA ATA CGA CTCACT ATA GGG AGA GGG AGG GAT GGA GCC TTT AAG GAG GTC AAA TGG CTA ACG (SEQID NO:1; the T7 promoter sequence is underlined, the bold G is +1 andthe bold C is a pause site at position +20); # T7phi17REV: GGC A-T CTAGA-TGC ATC CCT ATG CAG TCC TAA TGC (SEQ ID NO:2; contains Xba site);#F3: GGC AGC TAG CTA AAC ATG GCG CTG TAC GTT TCG C (SEQ ID NO:3;contains NheI restriction site at 5′ end); and #R3: AGC CTT TCG GAT CGAACA CGA TGA (SEQ ID NO:4).

The following table shows the reaction mixture used to prepare a 4.6 Kbfragment from T7 phage containing the T7 promoter. PCR amplification wasperformed under the following cycling conditions: 94° C. for 30″, 32cycles at 94° C. for 10″, 55° C. for 30″, 65° C. for 4′10″, 65° C. for10′, followed by a 4° C. hold.

Component Volume 5x LongAmp Buffer with Mg (New England 60 μl Biolabs)25 mM NTPs (each) 3.6 μl 10 mM # T7pPK13 12 μl (0.4 mM final) 10 mM#T7phi17REV 12 μl (0.4 mM final) (50 ng/μl) 6 μl H₂O 194.4 μl LongAmpPolymerase (NEB) 12 μl Total Reaction Volume 300 μl

The following table shows the reaction mixture used to prepare a 0.5 Kblambda fragment containing multiple biotins. PCR amplification wasperformed under the following cycling conditions: 94° C. for 10′, 32cycles at 94° C. for 10″, 55° C. for 30″, 72° C. for 1′, 72° C. for 7′,followed by a hold at 4° C.

Component Volume 10x TaqGold buffer w/o Mg (Applied Biosystems) 10 μl 10μM F3 6 μl 10 μM R3 6 μl 25 mM MgCl₂ 10 μl Lambda DNA (50 ng/μl) 2 μl 1mM dGTP 10 μl 1 mM dCTP 10 μl 1 mM dATP 10 μl 1 mM dTTP 6.5 μl 1 mMBio-16-dUTP 3.5 μl H₂O 21 μl TagGold Pol 5 μl Total Reaction Volume 100μl

Example 8 Single-Molecule Transcription and Sequencing Reaction

Condition 1 (Formation of Bi-Particles Inside the Flow Cell)

1000 μl of 1 micron Dynabeads MyOne Streptavidin T1 were diluted 1:100in PBS, pulled down by a magnet to wash, the supernatant was removed andthe beads were resuspended in 20 μl Buffer B+0.1% BSA. The beads weretransferred to a 0.5 ml tube and sonicated for 2 min before infusioninto the flow cell.

Non-magnetic polystyrene biotinylated 0.8 micron beads (Kisker,PC-B-0.8) were prepared as follow: 10 μl beads were spun down andresuspended in 10 μl Buffer B+0.1% BSA to produce a stock of washedbeads.

A PEG-Cu⁺⁺ functionalized glass slide (MicroSurfaces, Inc) waspassivated with Buffer B+1% BSA.

The following reaction was set up at room temperature and incubated for3 min at 37° C.

Component Volume 10x Buffer A 0.5 μl Template (5.1 kb PT7pK13-Bio DNA) 6ng/μl, 1.93 fmoles/μl, 2 μl or 2 nM (final 0.8 nM) 10x mix of three NTP(0.3 mM ATP + 0.3 mM GTP + 0.1 mM 1 μl UTP) 4 μM His-T7RNAP (final 0.8μM; prepared from stock by 1 μl diluting in Buffer A) H₂O 0.5 μl TotalReaction Volume 5 μl

45 μl of Buffer B was added to the reaction mix with T7 RNAP-DNAelongation complexes halted at position +20 of the template, and themixture was infused into the flow cell over a period of 5 min.

The flow cell was washed with Buffer B, and 1 μm SA magnetic beads (46μl Buffer B+0.1% BSA mixed with 6 μl washed beads in Buffer B+0.1% BSA)was infused over a period of 12 min. The flow cell was washed withBuffer B+0.1% BSA.

0.8 micron polystyrene biotinylated beads (2 μl of washed beads+48 μl1×B/0.1% BSA) were infused into the flow cell and incubated for 15 minto form bi-particles with surface tethered magnetic SA beads. The flowcell was washed with Buffer B to remove unbound 0.8 micron polystyrenebeads.

Transcription/sequencing was started by infusing Buffer B+250 μM NTPs+10mM DTT into the flow cell. Four different NTP mixes (each containingless of one of the nucleotides) were used in four different flow cells.

1x Buffer A 1x Buffer B 20 mM Tris pH 8.0 20 mM Tris pH 8.0 14 mM MgCl24 mM MgCl2 10 mM DTT 0.1 mM DTT 0.1 mM EDTA 0.1 mM EDTA 20 mM NaCl 20 mMNaCl 1.5% glycerol 20 μg/ml BSA 20 μg/ml BSA

Condition 2 (Pre-Formed Bi-Particles)

The overall transcription/sequencing reaction was set up as describedabove in Condition 1, but, instead of sequential deployment of magneticand polystyrene beads, the bi-particles were pre-formed as follow. 1000μl of the mix of 1 micron Dynabeads MyOne SA T1 (Dynal) diluted 1:100 inPBS, and 0.8 micron polystyrene biotin beads (Kisker) diluted 1:500,were mixed on a rotator for 30 min at room temperature to formbi-particles. The beads were pulled down with a magnet, the supernatantwith un-bound polystyrene particles was removed, and the beads werere-suspended in 20 μl of Buffer B+0.1% BSA. The beads were transferredto a 0.5 ml tube and sonicated for 2 min before adding to the reactionmix.

Example 9 Projected Cost for Whole Genome Sequencing UsingRotation-Dependent Transcriptional Sequencing

Significantly, one of the advantages of the rotation-dependenttranscriptional sequencing described herein is the low cost,particularly considering the very long run capability. The table belowshows the projected costs for sequencing the entire human genome.

Reagent (worth 100 flowchips) Cost DNA isolation (mini prep kit fromQiagen: can isolate more than   $2.45 1000 ng) DNA fragmentation(Invitrogen Nebulizer, small plastic each  $26.40 only NOT whole machine“Hydroshear”) DNA end-repair   $3.40 T4 DNA ligase   $1.50 Buffers  $2.00 SA paramagnetic beads 2.8 um (Dynal) $380.00 Biotinylated 0.9 umbeads (Spherotech)  $43.00 2 oligos for T7 promoter adaptor   $0.28 2PCR primers   $0.01 Biotin-labeling Kit   $1.12 Total cost for 100Flowchips $460.16 Total flowchips needed to sequence Whole Human genome16 Total cost per human genome  $73.63

It is to be understood that, while the systems, methods and compositionsof matter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the systems, methodsand compositions of matter. Other aspects, advantages, and modificationsare within the scope of the following claims.

Disclosed are systems, methods and compositions that can be used for,can be used in conjunction with, can be used in preparation for, or areproducts of the disclosed systems, methods and compositions. These andother materials are disclosed herein, and it is understood thatcombinations, subsets, interactions, groups, etc. of these systems,methods and compositions are disclosed. That is, while specificreference to each various individual and collective combinations andpermutations of these compositions and methods may not be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a particular system part, composition of matter orparticular method is disclosed and discussed and a number of systemparts, compositions or methods are discussed, each and every combinationand permutation of the system parts, compositions and methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

What is claimed is:
 1. A method of determining the sequence of a targetnucleic acid molecule having an unknown sequence, comprising: (a)combining an RNA polymerase, a target nucleic acid molecule, a solidsubstrate, and a rotation tag, to form a combination for sequencing inwhich the target nucleic acid molecule is bound to the rotation tag andthe RNA polymerase is immobilized on the solid substrate and complexedwith the target nucleic acid molecule; (b) exposing the combinationproduced in step (a) to sequencing conditions, wherein the sequencingconditions comprise contacting the combination of step (a) with at leastone nucleoside triphosphate; (c) detecting the rotational pattern of therotation tag; (d) repeating the contacting and detecting steps aplurality of times; and (e) determining the sequence of the targetnucleic acid molecule based, sequentially, on the presence or absence ofa change in the rotational pattern as a result of each exposure to thesequencing conditions.
 2. The method of claim 1, wherein step (a)comprises immobilizing the RNA polymerase on the solid substratefollowed by binding the rotation tag to the target nucleic acid moleculefollowed by complexing the rotation tagged-target nucleic acid moleculewith the immobilized RNA polymerase.
 3. The method of claim 1, whereinstep (a) comprises immobilizing the RNA polymerase on the solidsubstrate followed by complexing the target nucleic acid molecule withthe immobilized RNA polymerase followed by binding the rotation tag tothe target nucleic acid molecule.
 4. The method of claim 1, wherein step(a) comprises binding the rotation tag to the target nucleic acidmolecule followed by immobilizing the RNA polymerase on the solidsubstrate followed by complexing the rotation tagged-target nucleic acidmolecule with the immobilized RNA polymerase.
 5. The method of claim 1,wherein step (a) comprises binding the rotation tag to the targetnucleic acid molecules followed by complexing the rotation tagged-targetnucleic acid molecule with the RNA polymerase followed by immobilizingthe RNA polymerase of the complex on the solid substrate.
 6. The methodof claim 1, wherein step (a) comprises complexing the target nucleicacid molecule with the RNA polymerase followed by binding the rotationtag to the target nucleic acid molecule followed by immobilizing the RNApolymerase of the complex on the solid substrate.
 7. The method of claim1, wherein step (a) comprises complexing the target nucleic acidmolecule with the RNA polymerase followed by immobilizing the RNApolymerase of the complex on the solid substrate followed by binding therotation tag to the target nucleic acid molecule.
 8. The method of claim1, wherein the total diameter of the rotation tag is about 50 nm toabout 5 microns.
 9. The method of claim 8, wherein the rotation tagcomprises a single tag.
 10. The method of claim 8, wherein the rotationtag comprises a first tag and a second tag.
 11. The method of claim 10,wherein the first tag is about 2 microns in diameter.
 12. The method ofclaim 11, wherein the first tag and second tag are about the same size.13. The method of claim 10, wherein the first tag is larger than thesecond tag.
 14. The method of claim 10, wherein the first tag isattached to the target nucleic acid molecule and the second tag isattached to the first tag.
 15. The method of claim 10, wherein the firsttag is magnetic.
 16. The method of claim 10, wherein the second tag ismagnetic.
 17. The method of claim 10, wherein the second tag is anelectric tag.
 18. The method of claim 1, wherein the rotation tagcomprises a non-spherical tag.
 19. The method of claim 18, wherein thenon-spherical tag comprises a single tag.
 20. The method of claim 19,wherein the non-spherical tag has a shape selected from the groupconsisting of a tapered rod, triangular, conical, and egg-shaped. 21.The method of claim 18, wherein the non-spherical tag comprises a firsttag and a second tag.
 22. The method of claim 21, wherein the first tagis about 2 microns in diameter.
 23. The method of claim 22, wherein thefirst tag and second tag are about the same size.
 24. The method ofclaim 21, wherein the first tag is larger than the second tag.
 25. Themethod of claim 21, wherein the first tag is attached to the targetnucleic acid molecule and the second tag is attached to the first tag.26. The method of claim 21, wherein the first tag is magnetic.
 27. Themethod of claim 21, wherein the second tag is magnetic.
 28. The methodof claim 21, wherein the second tag is an electric tag.
 29. The methodof claim 1, wherein the rotation tag comprises a spherical tag.
 30. Themethod of claim 29, wherein the spherical tag comprises a single taghaving a non-uniform feature.
 31. The method of claim 30, wherein thenon-uniform feature is selected from the group consisting of shape,surface coating and material composition.
 32. The method of claim 29,wherein the tag comprises a first spherical tag and a second sphericaltag.
 33. The method of claim 32, wherein the first tag is about 2microns in diameter.
 34. The method of claim 33, wherein the first tagand second tag are about the same size.
 35. The method of claim 32,wherein the first tag is larger than the second tag.
 36. The method ofclaim 32, wherein the first tag is attached to the target nucleic acidmolecule and the second tag is attached to the first tag.
 37. The methodof claim 32, wherein the first tag is magnetic.
 38. The method of claim32, wherein the second tag is magnetic.
 39. The method of claim 32,wherein the second tag is an electric tag.
 40. The method of claim 1,wherein the rotation tag comprises at least a first tag, a second tag,and a third tag.
 41. The method of claim 1, wherein the RNA polymeraseis a multi-subunit RNA polymerase.
 42. The method of claim 1, whereinthe RNA polymerase is a single-subunit RNA polymerase.
 43. The method ofclaim 1, wherein the RNA polymerase exhibits a duration of rotation, pernucleotide incorporated, of from a sub-microsecond to about 100milliseconds.
 44. The method of claim 1, wherein the RNA polymeraseexhibits a duration of rotation, per nucleotide incorporated, of fromabout 100 milliseconds up to several seconds.